EP4362975A2 - Vaccins contre les sapovirus - Google Patents

Vaccins contre les sapovirus

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Publication number
EP4362975A2
EP4362975A2 EP22834024.6A EP22834024A EP4362975A2 EP 4362975 A2 EP4362975 A2 EP 4362975A2 EP 22834024 A EP22834024 A EP 22834024A EP 4362975 A2 EP4362975 A2 EP 4362975A2
Authority
EP
European Patent Office
Prior art keywords
antigens
sapovirus
seq
polypeptides
nucleic acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22834024.6A
Other languages
German (de)
English (en)
Inventor
Alan John YOUNG
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Vst dba Medgene Labs LLC
Original Assignee
Vst dba Medgene Labs LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Vst dba Medgene Labs LLC filed Critical Vst dba Medgene Labs LLC
Publication of EP4362975A2 publication Critical patent/EP4362975A2/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/12—Viral antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A61K2039/541—Mucosal route
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55566—Emulsions, e.g. Freund's adjuvant, MF59
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/16011—Caliciviridae
    • C12N2770/16022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/16011—Caliciviridae
    • C12N2770/16034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/16011—Caliciviridae
    • C12N2770/16051—Methods of production or purification of viral material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/16011—Caliciviridae
    • C12N2770/16071—Demonstrated in vivo effect

Definitions

  • the present invention pertains generally to compositions that elicit immune responses against Sapoviruses.
  • the invention relates to immunogenic compositions (e.g., vaccines) comprising immunogenic polypeptides of Sapovirus.
  • Immunogenic compositions may contain antigens other than Sapovirus antigens. Methods of eliciting an immune response with the immunogenic compositions as disclosed herein and methods of treating a Sapovirus infection are also described.
  • Sapoviruses are etiological agents of acute gastroenteritis. Sapoviruses are members of the Caliciviridae family of small, nonenveloped viruses, 27-35 nm in diameter, containing a single-strand of positive-sense genomic RNA.
  • Natural hosts for the virus are humans and swine. The virus is transmitted through oral/fecal contact. Symptoms most commonly include diarrhea and vomiting. The Sapovirus was initially discovered in an outbreak of gastroenteritis in an orphanage in Sapporo, Japan, 1977.
  • the present invention provides immunogenic compositions comprising Sapovirus antigens, in particular as a part of subunit vaccines.
  • Immunogenic compositions may include one or more polypeptides and/or adjuvants as described herein.
  • immunogenic compositions may comprise other antigens that may be used in immunization against pathogens that cause diarrheal diseases, such as antigens derived from rotavirus.
  • a process for producing a polypeptide including the step of culturing a host cell transformed with a nucleic acid as described herein under conditions which induce polypeptide expression.
  • a Sapovirus protein may be expressed by recombinant technology and used to develop an immunogenic composition comprising a recombinant antigenic subunit, where such expressed polypeptide is generated using baculovirus/insect cell methodology.
  • a process for producing nucleic acid where the nucleic acid encoding a Sapovirus derived protein or polypeptide is prepared (at least in part) by chemical synthesis.
  • the process includes amplifying nucleic acids using a primer-based amplification method (e.g., PCR).
  • the primers for such reactions include, but are not limited to, SEQ ID NOs: 3, 4, 5, 6, 9, 10, 11, 12, 13, 22, 23, 24, 25, and combinations thereof.
  • a process for producing a protein complex including administering a Sapovirus derived polypeptide, or a fragment thereof, to a subject.
  • the process includes admixing a Sapovirus-derived polypeptide with a pharmaceutically acceptable carrier or diluent.
  • the composition may include the polypeptide as set forth in SEQ ID NO:2, SEQ ID NO:8, SEQ ID NO: 15, SEQ ID NO:21 or a combination thereof.
  • the Sapovirus polypeptide comprises VPl.
  • the composition may contain more than one Sapovirus VP1, wherein the more than one VP Is are from more than one strain.
  • a method of eliciting an immunological response in a subject including administering a composition of the instant disclosure.
  • the method further includes administering an adjuvant.
  • the method includes administering the immunogenic composition to the subject via topical, parenteral or mucosal route.
  • the administration may be multiple administrations, where a first immunogenic composition and a second immunogenic composition are the same. In another aspect, the first immunogenic composition and the second immunogenic composition are different.
  • administration is performed two or more times.
  • a medicament for use in treating an infection by a Sapovirus in a subject in need thereof comprising a therapeutically effective amount of an immunogenic composition above is disclosed.
  • a method for treating an infection by a Sapovirus including administering to a subject in need thereof a therapeutically effective amount of an immunogenic composition as described herein.
  • the method includes mucosally administering a therapeutically effective amount of a first immunogenic composition comprising one or more Sapovirus antigens and topically or parenterally administering a therapeutically effective amount of a second immunogenic composition comprising one or more Sapovirus antigens.
  • an immunogenic composition comprises a rotavirus antigen.
  • an isolated recombinant nucleic acid encoding a Sapovirus VPl protein is disclosed, where the encoding nucleic acid includes i) a sequence including SEQ ID NO:l, SEQ ID NO:7, SEQ ID NO: 14, SEQ ID NO:20, and combinations thereof or ii) nucleic acid having at least 80% sequence identity to the nucleic acid sequences as set forth in SEQ ID NO: 1, SEQ ID NO: 7, SEQ ID NO: 14, SEQ ID NO:20 or combinations thereof.
  • the encoding nucleic acid is recombined with a vector.
  • the vector is a baculovirus vector.
  • the vector is contained in a host cell.
  • the host cell is an insect cell.
  • Figure 2 is a graph showing a serological assessment of vaccinates vs non-vaccinates via ELISA (same axis as noted in FIG. 1)( - ⁇ - 860 Vaccinate; 861 Vaccinate; - * - 862
  • Vaccinate - ⁇ - 863 Vaccinate; - A- 864 Vaccinate; 865 Vaccinate; -°- 866 Vaccinate; 867Non-Vacc; -Vacc; -x- 869Non-Vacc).
  • references to “a nucleic acid” includes one or more nucleic acids, and/or compositions of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.
  • compositions may "contain,” “comprise” or “consist essentially of' a particular component or group of components, where the skilled artisan would understand the latter to mean the scope of the claim is limited to the specified materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed invention.
  • Sapovirus refers to members of the genus Sapovirus of the family Caliciviridae of positive-sense, single-stranded RNA, nonenveloped viruses.
  • the term Sapovirus includes strains in all genogroups of the virus. Currently, Sapovirus strains are divided into five genogroups (GI-GV) based on their capsid (VP1) sequences.
  • the term Sapovirus may include, but is not limited to, the species Sapporo virus, London/29845 virus, Manchester virus, Houston/86 virus, Houston/90 virus, and Parkville virus. A large number of Sapovirus isolates have been partially or completely sequenced.
  • Sapovirus also includes isolates not characterized at the time of filing.
  • polypeptide and protein refer to a polymer of amino acid residues and are not limited to a minimum length of the product. Thus, peptides, oligopeptides, dimers, multimers, and the like, are included within the definition. Both full-length proteins and fragments thereof are encompassed by the definition.
  • the terms also include post-expression modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation and the like.
  • a "polypeptide” refers to a protein which includes modifications, such as deletions, additions and substitutions (generally conservative in nature), to the native sequence, so long as the protein maintains the desired activity. These modifications may be deliberate, as through site-directed mutagenesis, or may be accidental, such as through mutations of hosts which produce the proteins or errors due to PCR amplification.
  • substantially purified generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises the majority percent of the sample in which it resides.
  • a substantially purified component comprises about 50%, about 80%-85%, or about 90-95% of the sample.
  • Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.
  • isolated is meant, when referring to a polypeptide, that the indicated molecule is separate and discrete from the whole organism or cell with which the molecule is found in nature or is present in the substantial absence of other biological macro-molecules of the same type.
  • isolated with respect to a polynucleotide is a nucleic acid molecule devoid, in whole or part, of sequences normally associated with it in nature; or a sequence, as it exists in nature, but having heterologous sequences in association therewith; or a molecule disassociated from the chromosome.
  • label and “detectable label” refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin or haptens) and the like.
  • fluorescer refers to a substance or a portion thereof which is capable of exhibiting fluorescence in the detectable range.
  • labels include fluorescein, rhodamine, dansyl, umbelliferone, Texas red, luminol, acradimum esters, NADPH and a-b-galactosidase.
  • homologous refers to the percent identity between two polynucleotide or two polypeptide moieties.
  • Two nucleic acid, or two polypeptide sequences are "substantially homologous" to each other when the sequences exhibit at least about 50% sequence identity, at least about 75% sequence identity, at least about 80%-85% sequence identity, at least about 90% sequence identity, and at least about 95%-98% sequence identity over a defined length of the molecules.
  • substantially homologous also refers to sequences showing complete identity to the specified sequence.
  • antigens as disclosed herein may exclude naturally occurring Sapovirus sequences (i.e., amino acid sequences having 100% homology to the naturally occurring Sapovirus VP1 amino acid sequence or naturally occurring nucleic acid sequence encoding said amino acid sequence).
  • identity refers to an exact nucleotide-to-nucleotide or amino acid-to- amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity may be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs may be used to aid in the analysis, such as ALIGN, Dayhoff, M. O. in Atlas of Protein Sequence and Structure M. O. Dayhoff, ed., 5 Suppl.
  • nucleotide sequence identity is available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wis.) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence may be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.
  • Another method of establishing percent identity in the context of the present disclosure is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages the Smith-Waterman algorithm may be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the "Match" value reflects "sequence identity.”
  • Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters.
  • antigens as disclosed herein may exclude naturally occurring Sapovirus sequences (i.e., amino acid sequences having 100% identity to the naturally occurring Sapovirus VP1 amino acid sequence or naturally occurring nucleic acid sequence encoding said amino acid sequence).
  • homology may be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single-stranded-specific nuclease(s), and size determination of the digested fragments.
  • DNA sequences that are substantially homologous may be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art.
  • Recombinant as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature.
  • the term "recombinant” as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide.
  • the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.
  • transformation refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction or f-mating are included.
  • the exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.
  • Recombinant host cells refer to cells which may be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.
  • a "coding sequence” or a sequence which "encodes" a selected polypeptide is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or “control elements”).
  • the boundaries of the coding sequence may be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus.
  • a coding sequence may include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences.
  • a transcription termination sequence may be located 3' to the coding sequence.
  • control elements include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5' to the coding sequence), and translation termination sequences.
  • nucleic acid includes DNA and RNA, and also their analogues, such as those containing modified backbones (e.g., phosphorothioates, and the like), and also peptide nucleic acids (PNA), and the like.
  • PNA peptide nucleic acids
  • operably linked refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function.
  • a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present.
  • the promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof.
  • intervening untranslated yet transcribed sequences may be present between the promoter sequence and the coding sequence and the promoter sequence may still be considered “operably linked" to the coding sequence.
  • Encoded by refers to a nucleic acid sequence which codes for a polypeptide sequence, wherein the polypeptide sequence or a portion thereof contains an amino acid sequence of at least 3 to 5 amino acids, at least 8 to 10 amino acids, and at least 15 to 20 amino acids from a polypeptide encoded by the nucleic acid sequence.
  • Expression cassette or "expression construct” refers to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest.
  • An expression cassette generally includes control elements, as described above, such as a promoter which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well.
  • the expression cassette described herein may be contained within a plasmid construct.
  • the plasmid construct may also include, one or more selectable markers, a signal which allows the plasmid construct to exist as single-stranded DNA (e.g., a M13 origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).
  • a signal which allows the plasmid construct to exist as single-stranded DNA e.g., a M13 origin of replication
  • at least one multiple cloning site e.g., a SV40 or adenovirus origin of replication
  • Polynucleotide refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, less than about 70%, and less than about at least 90%, of the protein with which the polynucleotide is naturally associated.
  • Techniques for purifying polynucleotides of interest include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.
  • transfection is used to refer to the uptake of foreign DNA by a cell.
  • a cell has been "transfected” when exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are generally known in the art. Such techniques may be used to introduce one or more exogenous DNA moieties into suitable host cells.
  • the term refers to both stable and transient uptake of the genetic material, and includes uptake of peptide- or antibody-linked DNAs.
  • a “vector” is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes).
  • target cells e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes.
  • vector construct e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes.
  • expression vector e transfer vector
  • the term includes cloning and expression vehicles, as well as viral vectors.
  • fragment is intended a molecule consisting of only a part of the intact full-length sequence and structure.
  • a fragment of a polypeptide may include a C-terminal deletion, an N- terminal deletion, and/or an internal deletion of the native polypeptide.
  • a fragment of a polypeptide will generally include at least about 5-10 contiguous amino acid residues of the full- length molecule, at least about 15-25 contiguous amino acid residues of the full-length molecule, and at least about 20-50 or more contiguous amino acid residues of the full-length molecule, or any integer between 5 amino acids and the number of amino acids in the full-length sequence, provided that the fragment in question retains the ability to elicit the desired biological response.
  • a fragment of a nucleic acid may include a 5'-deletion, a 3'-deletion, and/or an internal deletion of a nucleic acid.
  • Nucleic acid fragments will generally include at least about 5-1000 contiguous nucleotide bases of the full-length molecule and may include at least 5, 10, 15, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides of the full-length molecule, or any integer between 5 nucleotides and the number of nucleotides in the full-length sequence.
  • Such fragments may be useful in hybridization, amplification, production of immunogenic fragments, or nucleic acid immunization.
  • immunogen a fragment of an immunogen which includes one or more epitopes and thus may modulate an immune response or may act as an adjuvant for a co-administered antigen.
  • fragments may be identified using any number of epitope mapping techniques, well known in the art. For example, linear epitopes may be determined by e.g., concurrently synthesizing large numbers of peptides on solid supports, the peptides corresponding to portions of the protein molecule, and reacting the peptides with antibodies while the peptides are still attached to the supports. Such techniques are known in the art and described in, e.g., U.S. Pat. No. 4,708,871, incorporated herein by reference in its entirety. Similarly, conformational epitopes are readily identified by determining spatial conformation of amino acids such as by, e.g., x-ray crystallography and 2-dimensional nuclear magnetic resonance.
  • Immunogenic fragments for purposes of the present disclosure, will usually be at least about 2 amino acids in length, about 5 amino acids in length, and at least about 10 to about 15 amino acids in length. There is no critical upper limit to the length of the fragment, which could comprise nearly the full-length of the protein sequence, or even a fusion protein comprising two or more epitopes.
  • epitope generally refers to the site on an antigen which is recognized by a T-cell receptor and/or an antibody. In embodiments, it is a short peptide derived from or as part of a protein antigen. However, the term is also intended to include peptides with glycopeptides and carbohydrate epitopes. Several different epitopes may be carried by a single antigenic molecule. The term “epitope” also includes modified sequences of amino acids or carbohydrates which stimulate responses which recognize the whole organism. It is advantageous if the selected epitope is an epitope of an infectious agent, which agent causes the infectious disease.
  • the epitope may be generated from knowledge of the amino acid and corresponding DNA sequences of the peptide or polypeptide, as well as from the nature of particular amino acids (e.g., size, charge, and the like) and the codon dictionary, without undue experimentation.
  • Some guidelines in determining whether a protein will stimulate a response include: Peptide length — the peptide is about 8 or 9 amino acids long to fit into the MHC class I complex and about 13-25 amino acids long to fit into a class II MHC complex. This length is a minimum for the peptide to bind to the MHC complex.
  • the peptides may be longer than these lengths because cells may cut peptides.
  • the peptide may contain an appropriate anchor motif which will enable it to bind to the various class I or class II molecules with high enough specificity to generate an immune response. This may be done, without undue experimentation, by comparing the sequence of the protein of interest with published structures of peptides associated with the MHC molecules. Thus, the skilled artisan may ascertain an epitope of interest by comparing the protein sequence with sequences listed in the protein database.
  • T cell epitope refers generally to those features of a peptide structure which are capable of inducing a T cell response and a "B cell epitope” refers generally to those features of a peptide structure which are capable of inducing a B cell response.
  • An "immunological response" to an antigen or composition is the development in a subject of a humoral and/or a cellular immune response to an antigen present in the composition of interest.
  • a “humoral immune response” refers to an immune response mediated by antibody molecules
  • a “cellular immune response” is one mediated by T-lymphocytes and/or other white blood cells.
  • CTL cytolytic T-cells
  • CTLs have specificity for peptide antigens that are presented in association with proteins encoded by the major histocompatibility complex (MHC) and expressed on the surfaces of cells.
  • MHC major histocompatibility complex
  • helper T-cells help induce and promote the destruction of intracellular microbes, or the lysis of cells infected with such microbes.
  • Another aspect of cellular immunity involves an antigen-specific response by helper T-cells.
  • Helper T-cells act to help stimulate the function, and focus the activity of, nonspecific effector cells against cells displaying peptide antigens in association with MHC molecules on their surface.
  • a "cellular immune response” also refers to the production of cytokines, chemokines and other such molecules produced by activated T-cells and/or other white blood cells, including those derived from CD4+ and CD8+ T-cells.
  • a composition or vaccine that elicits a cellular immune response may serve to sensitize a vertebrate subject by the presentation of antigen in association with MHC molecules at the cell surface.
  • the cell-mediated immune response is directed at, or near, cells presenting antigen at their surface.
  • antigen-specific T-lymphocytes may be generated to allow for the future protection of an immunized host.
  • the ability of a particular antigen to stimulate a cell-mediated immunological response may be determined by a number of assays, such as by lymphoproliferation (lymphocyte activation) assays, CTL cytotoxic cell assays, or by assaying for T-lymphocytes specific for the antigen in a sensitized subject. Such assays are well known in the art. Recent methods of measuring cell-mediated immune response include measurement of intracellular cytokines or cytokine secretion by T-cell populations, or by measurement of epitope specific T-cells.
  • an immunological response as used herein may be one that stimulates the production of antibodies (e.g., neutralizing antibodies that block bacterial toxins and pathogens such as viruses entering cells and replicating by binding to toxins and pathogens, typically protecting cells from infection and destruction).
  • the antigen of interest may also elicit production of CTLs.
  • an immunological response may include one or more of the following effects: the production of antibodies by B-cells; and/or the activation of suppressor T-cells and/or memory/effector T-cells directed specifically to an antigen or antigens present in the composition or vaccine of interest.
  • responses may serve to neutralize infectivity, and/or mediate antibody-complement, or antibody dependent cell cytotoxicity (ADCC) to provide protection to an immunized host.
  • ADCC antibody dependent cell cytotoxicity
  • Such responses may be determined using standard immunoassays and neutralization assays, well known in the art.
  • the innate immune system of mammals also recognizes and responds to molecular features of pathogenic organisms via activation of Toll-like receptors and similar receptor molecules on immune cells. Upon activation of the innate immune system, various non-adaptive immune response cells are activated to, e.g., produce various cytokines, lymphokines and chemokines.
  • Cells activated by an innate immune response include immature and mature Dendritic cells of the moncyte and plamsacytoid lineage (MDC, PDC), as well as gamma, delta, alpha and beta T cells and B cells and the like.
  • MDC moncyte and plamsacytoid lineage
  • gamma, delta, alpha and beta T cells and B cells and the like are immature and mature Dendritic cells of the moncyte and plamsacytoid lineage (MDC, PDC), as well as gamma, delta, alpha and beta T cells and B cells and the like.
  • An "immunogenic composition” is a composition that comprises an antigenic molecule where administration of the composition to a subject results in the development in the subject of a humoral and/or a cellular immune response to the antigenic molecule of interest.
  • immunogenic protein or polypeptide refer to an amino acid sequence which elicits an immunological response as described above.
  • An "immunogenic" protein or polypeptide, as used herein, includes the full-length sequence of the protein in question, including the precursor and mature forms, analogs thereof, or immunogenic fragments thereof.
  • Gene transfer refers to methods or systems for reliably inserting DNA or RNA of interest into a host cell. Such methods may result in transient expression of non- integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells.
  • Gene delivery expression vectors include, but are not limited to, vectors derived from bacterial plasmid vectors, viral vectors, non-viral vectors, alphaviruses, pox viruses and vaccinia viruses. When used for immunization, such gene delivery expression vectors may be referred to as vaccines or vaccine vectors.
  • derived from is used herein to identify the original source of a molecule but is not meant to limit the method by which the molecule is made which may be, for example, by chemical synthesis or recombinant means.
  • a viral polypeptide is "derived from" a particular polypeptide of a virus (viral polypeptide) if it is (i) encoded by an open reading frame of a polynucleotide of that virus (viral polynucleotide), or (ii) displays sequence identity to polypeptides of that virus as described above.
  • a polynucleotide "derived from" a designated sequence refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least about 6 nucleotides, at least about 8 nucleotides, at least about 10-12 nucleotides, and at least about 15-20 nucleotides corresponding, i.e., identical or complementary to, a region of the designated nucleotide sequence.
  • the derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.
  • a Sapovirus polynucleotide, oligonucleotide, nucleic acid, protein, polypeptide, or peptide, as defined above, is a molecule derived from a Sapovirus, respectively, including, without limitation, any of the various isolates of Sapovirus.
  • the molecule need not be physically derived from the particular isolate in question, but may be synthetically or recombinantly produced.
  • the genomes of Sapovirus strains contain either two or three open reading frames.
  • ORF1 encodes a polyprotein comprising both nonstructural and structural proteins.
  • the capsid protein VP1 is encoded by ORF1 as a component of the Sapovirus polyprotein, and the minor structural protein VP10 is encoded by ORF2.
  • a stop codon precedes the coding region for the capsid protein.
  • a polyprotein not including the capsid protein is encoded by ORF1
  • the capsid protein VP1 is encoded by ORF2
  • the minor structural protein VP10 is encoded by ORF3.
  • Cleavage of the Sapovirus strain Me 10 polyprotein (GenBank Accession No. AY237420) by a 3C-like protease produces at least ten distinct products, pi 1, p28, p35 (NTPase), p32, pl4 (VPg), p70 (Pro-Pol), p60 (VP1).
  • the polyprotein comprises the polypeptides in the order of NH 2 -pll-p28-NTPase-p32-VPg-p70(Pro-Pol)-VPl-COOH.
  • the p70 (Pro-Pol) region of the polyprotein resides at residues 1056-1720, and the VP1 region of the polyprotein resides at residues 1721-2278.
  • Nucleic acid and protein sequences for a number of Sapovirus isolates are also known. Representative Sapovirus sequences are presented in SEQ ID NOS:l, 7, 14, and 20. Additional representative sequences, including sequences of ORFl and ORF2, and their encoded polypeptides from Sapovirus isolates are listed in the National Center for Biotechnology Information (NCBI) database. See, for example, GenBank entries: Sapovirus MclO, GenBank Accession No. NC ⁇ 010624; Sapporo virus, GenBank Accession No. U65427; Sapovirus MclO, GenBank Accession No. AY237420; Sapovirus SaKaeo- 15/Thailand, GenBank Accession No.
  • NCBI National Center for Biotechnology Information
  • capsid protein or “capsid polypeptide” or “VP1” in reference to a Sapovirus refer to a polypeptide comprising a sequence homologous or identical to the capsid polypeptide of a Sapovirus, and include sequences displaying at least about 80-99.9% sequence identity thereto, including any percent identity within these ranges, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% sequence identity thereto.
  • the capsid polypeptide may be encoded by either ORF1 or ORF2 in different strains of Sapovirus.
  • the Sapovirus has two open reading frames: the capsid protein is encoded by ORF1 as part of a polyprotein and a minor structural protein (VP 10) is encoded by ORF2.
  • the Sapovirus has three open reading frames: a stop codon precedes the coding region for the capsid protein, which is encoded by ORF2, and a minor structural protein (VP 10) is encoded by ORF3.
  • minor structural protein or “minor structural polypeptide” or “VP 10" in reference to a Sapovirus refer to a polypeptide comprising a sequence homologous or identical to the polypeptide encoded by the open reading frame following the coding region for the capsid protein in the Sapovirus genome (either ORF2 or ORF3 depending on the strain), and include sequences displaying at least about 80-100% sequence identity thereto, including any percent identity within these ranges, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99.9% sequence identity thereto.
  • Sapovirus polyprotein refers to a polyprotein comprising a sequence homologous or identical to the ORF1 -encoded polyprotein of a Sapovirus, and includes sequences displaying at least about 80-100% sequence identity thereto, including any percent identity within these ranges, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.9% sequence identity thereto.
  • an "antigen” refers to a molecule containing one or more epitopes (either linear, conformational or both) that will stimulate a host's immune-system to make a humoral and/or cellular antigen-specific response.
  • the term is used interchangeably with the term "immunogen.”
  • a B-cell epitope will include at least about 5 amino acids but may be as small as 3-4 amino acids.
  • AT-cell epitope, such as a CTL epitope will include at least about 7-9 amino acids, and a helper T-cell epitope at least about 12-20 amino acids.
  • an epitope will include between about 7 and 15 amino acids, such as, 9, 10, 12 or 15 amino acids.
  • antigen denotes both subunit antigens (i.e., antigens which are separate and discrete from a whole organism with which the antigen is associated in nature), as well as, killed, attenuated or inactivated bacteria, viruses, fungi, parasites or other microbes.
  • Antibodies such as anti-idiotype antibodies, or fragments thereof, and synthetic peptide mimotopes, which may mimic an antigen or antigenic determinant, are also captured under the definition of antigen as used herein.
  • an oligonucleotide or polynucleotide which expresses an antigen or antigenic determinant in vivo, such as in gene therapy and DNA immunization applications, is also included in the definition of antigen herein.
  • antibody encompasses polyclonal and monoclonal antibody preparations, as well as preparations including hybrid antibodies, altered antibodies, chimeric antibodies and, humanized antibodies, as well as: hybrid (chimeric) antibody molecules and any functional fragments obtained from such molecules, wherein such fragments retain specific-binding properties of the parent antibody molecule.
  • hybridize and “hybridization” refer to the formation of complexes between nucleotide sequences which are sufficiently complementary to form complexes via Watson-Crick base pairing.
  • target template
  • such complexes (or hybrids) are sufficiently stable to serve the priming function required by, e.g., the DNA polymerase to initiate DNA synthesis.
  • a "biological sample” refers to a sample of tissue or fluid isolated from a subject, including but not limited to, for example, blood, plasma, serum, fecal matter, urine, bone marrow, bile, spinal fluid, lymph fluid, samples of the skin, external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, organs, biopsies and also samples of in vitro cell culture constituents including but not limited to conditioned media resulting from the growth of cells and tissues in culture medium, e.g., recombinant cells, and cell components.
  • Sapovirus may be obtained from biological samples such as vomit or diarrhea from individuals infected with the viruses.
  • subject any member of the subphylum chordata, including, without limitation, humans and other primates, including non-human primates such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats, deer and horses; domestic mammals such as dogs and cats; laboratory animals including rodents such as mice, rats and guinea pigs; birds, including domestic, wild and game birds such as chickens, turkeys and other gallinaceous birds, ducks, geese, wild and game animals, and the like.
  • the term does not denote a particular age. Thus, both adult and newborn individuals are intended to be covered.
  • variant refers to biologically active derivatives of the reference molecule that retain desired activity, such as antigenic activity in inducing an immune response against Sapovirus.
  • analog refers to compounds having a native polypeptide sequence and structure with one or more amino acid additions, substitutions (generally conservative in nature) and/or deletions, relative to the native molecule, so long as the modifications do not destroy biological activity and which are "substantially homologous" to the reference molecule as defined below.
  • amino acid sequences of such analogs will have a high degree of sequence homology to the reference sequence, e.g., amino acid sequence homology of more than 50%, generally more than 60%- 70%, even more particularly 80%-85% or more, such as at least 90%-95% or more, when the two sequences are aligned.
  • the analogs will include the same number of amino acids but will include substitutions, as explained herein.
  • mutant further includes polypeptides having one or more amino acid-like molecules including but not limited to compounds comprising only amino and/or imino molecules, polypeptides containing one or more analogs of an amino acid (including, for example, unnatural amino acids, and the like), polypeptides with substituted linkages, as well as other modifications known in the art, both naturally occurring and non-naturally occurring (e.g., synthetic), cyclized, branched molecules and the like.
  • the term also includes molecules comprising one or more N-substituted glycine residues (a "peptoid") and other synthetic amino acids or peptides. (See, e.g., U.S. Pat. Nos.
  • the analog or mutein has at least the same antigenic activity as the native molecule.
  • Methods for making polypeptide analogs and muteins are known in the art and are described further below.
  • analogs generally include substitutions that are conservative in nature, i.e., those substitutions that take place within a family of amino acids that are related in their side chains.
  • amino acids are generally divided into four families: (1) acidic— aspartate and glutamate; (2) basic— lysine, arginine, histidine; (3) non-polar— alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan; and (4) uncharged polar- glycine, asparagine, glutamine, cysteine, serine threonine, tyrosine.
  • Phenylalanine, tryptophan, and tyrosine are sometimes classified as aromatic amino acids.
  • the polypeptide of interest may include up to about 5-10 conservative or non conservative amino acid substitutions, or even up to about 15-25 conservative or non conservative amino acid substitutions, or any integer between 5-25, so long as the desired function of the molecule remains intact.
  • One of skill in the art may readily determine regions of the molecule of interest that may tolerate change by reference to Hopp/ Woods and Kyte-Doolittle plots, well known in the art.
  • multiple epitope fusion antigen or “multiple epitope fusion protein” as used herein intends a polypeptide in which multiple Sapovirus antigens are part of a single, continuous chain of amino acids, which chain does not occur in nature.
  • the Sapovirus antigens may be connected directly to each other by peptide bonds or may be separated by intervening amino acid sequences.
  • the fusion antigens may contain ORF1 -encoded, ORF2-encoded, and/or ORF3- encoded polypeptides or fragments thereof, including, for example, sequences of Sapovirus polypeptides, such as N-terminal protein, pi 1, p28, NTPase, p32, VPg, protease, polymerase, VP1, and VP10.
  • the fusion antigens may also contain sequences exogenous to the Sapovirus. Moreover, the sequences present may be from multiple genotypes and/or isolates of Sapovirus.
  • terapéuticaally effective amount in the context of the immunogenic compositions is meant an amount of an immunogen (e.g., immunogenic polypeptide, fusion protein, polyprotein, or nucleic acid encoding an antigen) which will induce an immunological response, either for antibody production or for treatment or prevention of Sapovirus infection. Such a response will generally result in the development in the subject of an antibody-mediated and/or a secretory or cellular immune response to the composition.
  • an immunogen e.g., immunogenic polypeptide, fusion protein, polyprotein, or nucleic acid encoding an antigen
  • such a response includes but is not limited to one or more of the following effects; the production of antibodies from any of the immunological classes, such as immunoglobulins A, D, E, G or M; the proliferation of B and T lymphocytes; the provision of activation, growth and differentiation signals to immunological cells; expansion of helper T cell, suppressor T cell, and/or cytotoxic T cell and/or g, d-T cell populations.
  • immunological classes such as immunoglobulins A, D, E, G or M
  • B and T lymphocytes the proliferation of B and T lymphocytes
  • the provision of activation, growth and differentiation signals to immunological cells expansion of helper T cell, suppressor T cell, and/or cytotoxic T cell and/or g, d-T cell populations.
  • an "effective amount" of an adjuvant will be that amount which enhances an immunological response to a co-administered antigen or nucleic acid encoding an antigen.
  • treatment refers to any of (i) the prevention of infection or reinfection, as in a traditional vaccine, (ii) the reduction or elimination of symptoms, and (iii) the substantial or complete elimination of the pathogen in question. Treatment may be effected prophylactically (prior to infection) or therapeutically (following infection).
  • the present disclosure includes compositions and methods for immunizing a subject against Sapovirus infection.
  • the instant disclosure provides immunogenic compositions comprising nucleic acids encoding capsid proteins and/or other immunogenic polypeptides from one or more strains of Sapovirus, compositions comprising immunogenic polypeptides derived from one or more strains of Sapovirus.
  • Immunogenic polypeptides to be used in the practice of the instant subject matter may include Sapovirus-derived polypeptides, including ORF1 -encoded polypeptides, ORF2-encoded polypeptides, ORF3 -encoded polypeptides, multiple epitope fusion antigens, and/or ORF1 -encoded polyproteins.
  • immunogenic compositions may comprise one or more adjuvants or nucleic acids encoding adjuvants, wherein immunogenic polypeptides are mixed or co-expressed with adjuvants.
  • Immunogenic compositions may also comprise additional antigens other than Sapovirus antigens, such as antigens that may be used in immunization against pathogens that cause diarrheal diseases.
  • nucleic acids and polypeptides for use in immunogenic compositions and methods of using such compositions in the treatment or prevention of Sapovirus infection.
  • the immunogenic compositions described herein may comprise one or more polypeptides derived from one or more genotypes and/or isolates of Sapovirus.
  • Polypeptides that may be used in the practice of the subject matter as disclosed herein include structural proteins, nonstructural proteins, and polyproteins. Such polypeptides may be full-length proteins or variants or immunogenic fragments thereof capable of eliciting an immune response to a Sapovirus.
  • Cleavage of the Sapovirus strain MclO polyprotein, for example (GenBank Accession No. AY237420), by a 3C-like protease produces at least ten distinct products, pi 1, p28, p35 (NTPase), p32, pl4 (VPg), p70 (Pro-Pol), p60 (VP1).
  • Initial proteolytic processing produces p66 (p28-p35), p46 (p32-pl4), and pl20 (p32-pl4-p70) fragments.
  • the polyprotein comprises the polypeptides in the order of NH 2 --pll-p28-NTPase-p32-VPg-p70(Pro-Pol)-VPl-COOH.
  • the p70 (Pro-Pol) region of the polyprotein resides at residues 1056-1720, and the VP1 region of the polyprotein resides at residues 1721-2278 (numbered relative to GenBank Accession No. AY237420).
  • Nucleic acid and amino acid sequences of a number of Sapovirus strains and isolates including nucleic acid and amino acid sequences of VP1 and VP 10 structural proteins and the various regions of Sapovirus polyproteins, including pi 1, p28, NTPase, p32, VPg, p70(Pro-Pol), VPl genes and polypeptides have also been determined.
  • the polypeptides in immunogenic compositions may be encoded by any region of a Sapovirus genome. Multiple polypeptides may be included in immunogenic compositions. Such compositions may comprise polypeptides from the same Sapovirus isolate or from different strains and isolates, including isolates having any of the various Sapovirus genotypes, to provide increased protection against a broad range of Sapovirus genotypes. Multiple viral strains of Sapovirus are known, and multiple polypeptides comprising epitopes derived from any of these strains may be used in immunogenic compositions.
  • the antigens used in the immunogenic compositions of the present disclosure may be present in the composition as individual separate polypeptides.
  • the recombinant proteins of the present disclosure are expressed as a GST-fusion protein and/or a His-tagged fusion protein.
  • the immunogenic compositions described herein may also comprise multiple epitope fusion proteins. See, e.g., International Publication No. WO 97/44469, U.S. Pat. No. 6,632,601, U.S. Pat. No. 6,630,298, U.S. Pat. No. 6,514,731, and U.S. Pat. No. 6,797,809; herein incorporated by reference in their entireties.
  • Such fusion proteins include multiple epitopes derived from two or more viral polypeptides of one or more genotypes and/or isolates of Sapovirus.
  • Multiple epitope fusion proteins offer two principal advantages: first, a polypeptide that may be unstable or poorly expressed on its own may be assisted by adding a suitable hybrid partner that overcomes the problem; second, commercial manufacture is simplified as only one expression and purification need be employed in order to produce two polypeptides which are both antigenically useful.
  • Multiepitope fusion proteins may contain one or more of the various domains of Sapovirus polyproteins, full-length polyproteins, VP1 (also referred to herein as a capsid protein) and/or VP 10 (also referred to herein as a Sapovirus minor structural protein); or fragments thereof, derived from one or more Sapovirus isolates.
  • the polypeptides in fusion proteins may be derived from the same Sapovirus isolate or from different strains and isolates, including isolates having any of the various Sapovirus genotypes, to provide increased protection against a broad range of Sapovirus genotypes. Multiple viral strains of Sapovirus are known, and epitopes derived from any of these strains may be used in a fusion protein.
  • Sapovirus includes at least five genogroups (Gl-GV). Each strain includes a number of antigenic determinants that are in homologous regions present in all strains of Sapoviruses but are slightly different from one viral strain to another.
  • a multiple epitope fusion antigen may include multiple polypeptides from different viral strains of Sapovirus, each comprising a particular homologous region but each having a different form of an antigenic determinant.
  • antigenic determinants may have a high degree of homology in terms of amino acid sequence, which degree of homology is generally 30% or more, 40% or more, when aligned.
  • a fusion protein may also comprise multiple copies of an epitope, wherein one or more polypeptides of the fusion protein comprise sequences comprising exact copies of the same epitope.
  • polypeptides may be selected based on the particular viral clades endemic in specific geographic regions where vaccine compositions containing the fusions will be used. It is readily apparent that the subject fusions provide an effective means of treating Sapovirus infection in a wide variety of contexts.
  • Multiple epitope fusion antigens may be represented by the formula NH2-A- ⁇ -X-L- ⁇ n - B— COOH, wherein: X is an amino acid sequence of a Sapovirus antigen or a fragment thereof; L is an optional linker amino acid sequence; A is an optional N-terminal amino acid sequence; B is an optional C-terminal amino acid sequence; and n is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.
  • an —X— moiety has a leader peptide sequence in its wild-type form, this may be included or omitted in the multiple epitope fusion antigen.
  • the leader peptides will be deleted except for that of the —X— moiety located at the N-terminus of the hybrid protein i.e., the leader peptide of Xi will be retained, but the leader peptides of X2 . . . X n will be omitted. This is equivalent to deleting all leader peptides and using the leader peptide of Xi as moiety -A-.
  • linker amino acid sequence -L- may be present or absent.
  • the hybrid may be NH2— X1-L1-X2-L2-COOH, NH2--X1--X2-- COOH, NH2-X1-L1-X2-COOH, NH2-X1-X2-L2-COOH, and the like.
  • Linker amino acid sequence(s)-L- will typically be short, e.g., 20 or fewer amino acids (i.e., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1).
  • Other suitable linker amino acid sequences will be apparent to those skilled in the art.
  • a useful linker is GSGGGG, with the Gly- Ser dipeptide being formed from a BamHI restriction site, which aids cloning and manipulation, and the (Gly) 4 tetrapeptide being a typical poly-glycine linker.
  • protease substrate sequences may also be added (e.g., TEV protease: ENLYFQG).
  • -A- is an optional N-terminal amino acid sequence. This will typically be short, e.g.,
  • 40 or fewer amino acids i.e., 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23,
  • Other suitable N- terminal amino acid sequences will be apparent to those skilled in the art. If Xi lacks its own N- terminus methionine, -A- is an oligopeptide (e.g., with 1, 2, 3, 4, 5, 6, 7 or 8 amino acids) which provides a N-terminus methionine.
  • B— is an optional C-terminal amino acid sequence. This will typically be short, e.g.,
  • 40 or fewer amino acids i.e., 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23,
  • Other suitable C-terminal amino acid sequences will be apparent to those skilled in the art, including that such His n sequences may be removed when a TEV protease substrate sequence precedes it (e.g., ENLYFQGHis n ).
  • antigenic determinants from different Sapovirus strains may be present.
  • Representative multiepitope fusion proteins for use in the present disclosure, comprising polypeptides derived from Sapovirus isolates, are discussed below. However, it is to be understood that multiepitope fusion proteins comprising other epitopes derived from Sapovirus genomes or multiepitope fusion proteins comprising different arrangements of epitopes will also find use in immunogenic compositions as disclosed.
  • the fusion protein comprises one or more capsid and/or minor structural polypeptides from one or more isolates of Sapovirus.
  • the fusion protein comprises VP1 polypeptides from more than one Sapovirus strain (e.g., VPlsapporo-VPlLondon/29845, VPlLondon/29845-VPlManchester- VP 1 Sapporo, VP 1 Manchester- VP lparkville-VP 1 Sapporo-VP lLondon/29845, VP lparkville-VP lHouston/90- VP 1 Houston/86- VP 1 Manchester- VP 1 Sapporo) ⁇
  • Sapovirus strain e.g., VPlsapporo-VPlLondon/29845, VPlLondon/29845-VPlManchester- VP 1 Sapporo, VP 1 Manchester- VP lparkville-VP 1 Sapporo-VP lLondon/29845, VP lparkville-VP lHouston/90- VP 1 Houston/86- VP 1 Manchester- VP 1 Sapporo
  • the viral regions need not be in the order in which they occur naturally. Moreover, each of the regions may be derived from the same or different Sapovirus isolates.
  • the various Sapovirus polypeptides present in the various fusions described above may either be full-length polypeptides or portions thereof.
  • the fusion proteins also may contain other amino acid sequences, such as amino acid linkers or signal sequences, as well as ligands useful in protein purification, such as glutathione-S-transferase and staphylococcal protein A.
  • Nucleic acids for use as disclosed herein may be derived from any of the various regions of a Sapovirus genome, including from any of the ORF1, ORF2, or ORF3 regions. Representative sequences from Sapovirus isolates are listed herein. Thus, nucleic acids for use as disclosed herein include those derived from one or more sequences from any pathogenic Sapovirus genotype or isolate.
  • Representative sequences from Sapovirus are known, including SEQ ID NOs:l, 7, 14, and 20.
  • Representative Sapovirus sequences are Sapporo virus-London/29845, GenBank Accession No. U95645, Parkville virus, GenBank Accession No. AF294739; and Sapporo virus- Houston/86, GenBank Accession No. U95643.
  • sequences Any of these sequences, as well as fragments and variants thereof that may be used in nucleic acid immunization to elicit an immune response to a Sapovirus will find use in the present methods.
  • the present disclosure provides variants of the above sequences displaying at least about 80-100% sequence identity thereto, including any percent identity within these ranges, such as 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.9% sequence identity thereto.
  • the present disclosure also provides polynucleotides encoding immunogenic fragments of a Sapovirus polypeptide derived from any of the above sequences or a variant thereof.
  • Polynucleotides may also comprise coding sequences for polypeptides which occur naturally or may be artificial sequences which do not occur in nature.
  • Polynucleotides may contain less than an entire Sapovirus genome, or alternatively may include the sequence of an entire viral genomic RNA.
  • polynucleotides may comprise one or more sequences from the ORFl, ORF2, and ORF3 regions of a Sapovirus genome.
  • Polynucleotides may also comprise the entire viral genomic RNA or less than the entire viral genomic RNA from multiple genotypes and/or isolates of Sapovirus.
  • polynucleotides comprise an ORFl sequence coding for the full- length polyprotein of a Sapovirus. In other embodiments, polynucleotides comprise one or more portions of the ORFl sequence of a Sapovirus.
  • a polynucleotide may comprise a nucleotide sequence encoding a portion of a Sapovirus polyprotein.
  • the polynucleotides comprise sequences encoding one or more capsid proteins of a Sapovirus.
  • polynucleotides may comprise one or more sequences coding for structural proteins (e.g., VP1, VP2, VP10) of a Sapovirus.
  • polynucleotides comprise sequences coding for at least two capsid proteins from multiple genotypes and/or isolates of Sapovirus.
  • polynucleotides comprise one or more Sapovirus sequences coding for the capsid proteins of one or more isolates of Sapovirus. In certain embodiments, polynucleotides comprise one or more sequences coding for the capsid proteins of one or more isolates of Sapovirus.
  • Multiepitope fusion proteins may comprise sequences from one or more genotypes and/or isolates of Sapovirus.
  • Nucleic acids according to the instant disclosure may be prepared in many ways (e.g., by chemical synthesis, from genomic or cDNA libraries, from the organism itself, etc.) and may take various forms (e.g., single stranded, double stranded, vectors, probes, and the like). In embodiments, nucleic acids are prepared in substantially pure form (i.e., substantially free from other host cell or non-host cell nucleic acids).
  • nucleic acids may be obtained by screening cDNA and/or genomic libraries from cells infected with virus, or by deriving the gene from a vector known to include the same.
  • polynucleotides of interest may be isolated from a genomic library derived from viral RNA, present in, for example, stool or vomit samples from an infected individual.
  • Sapovirus nucleic acids may be isolated from infected humans or other mammals or from stool or vomit samples collected from infected individuals.
  • Viruses may be grown in LLC-PK cells in the presence of intestinal fluid containing bile acids.
  • An amplification method such as PCR may be used to amplify polynucleotides from either Sapovirus genomic RNA or cDNA encoding therefor.
  • polynucleotides may be synthesized in the laboratory, for example, using an automatic synthesizer.
  • the nucleotide sequence may be designed with the appropriate codons for the particular amino acid sequence desired. In general, one will select preferred codons for the intended host in which the sequence will be expressed.
  • the complete sequence of the polynucleotide of interest may be assembled from overlapping oligonucleotides prepared by standard methods and assembled into a complete coding sequence.
  • the polynucleotides may be RNA or single- or double-stranded DNA. In embodiments, the polynucleotides are isolated free of other components, such as proteins and lipids.
  • nucleotide sequences may be obtained from vectors harboring the desired sequences or synthesized completely or in part using various oligonucleotide synthesis techniques known in the art, such as site-directed mutagenesis and polymerase chain reaction (PCR) techniques where appropriate.
  • oligonucleotide synthesis techniques such as site-directed mutagenesis and polymerase chain reaction (PCR) techniques where appropriate.
  • PCR polymerase chain reaction
  • one method of obtaining nucleotide sequences encoding the desired sequences is by annealing complementary sets of overlapping synthetic oligonucleotides produced in a conventional, automated polynucleotide synthesizer, followed by ligation with an appropriate DNA ligase and amplification of the ligated nucleotide sequence via PCR.
  • Polypeptides described herein may be prepared in any suitable manner (e.g., recombinant expression, purification from cell culture, chemical synthesis, and the like) and in various forms (e.g., native, fusions, non-glycosylated, lipidated, and the like). Such polypeptides include naturally-occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art. Polypeptides are prepared in substantially pure form (i.e., substantially free from other host cell or non-host cell proteins).
  • Polypeptides may be conveniently synthesized chemically, by any of several techniques that are known to those skilled in the peptide art. In general, these methods employ the sequential addition of one or more amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected or derivatized amino acid may then be either attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected, under conditions that allow for the formation of an amide linkage. The protecting group is then removed from the newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth.
  • any remaining protecting groups and any solid support, if solid phase synthesis techniques are used are removed sequentially or concurrently, to render the final polypeptide.
  • this general procedure it is possible to add more than one amino acid at a time to a growing chain, for example, by coupling (under conditions which do not racemize chiral centers) a protected tripeptide with a properly protected dipeptide to form, after deprotection, a pentapeptide.
  • Typical protecting groups include t-butyloxy carbonyl (Boc), 9- fluorenylmethoxycarbonyl (Fmoc) benzyloxycarbonyl (Cbz); p-toluenesulfonyl (Tx); 2,4- dinitrophenyl; benzyl (Bzl); biphenylisopropyloxycarboxy-carbonyl, t-amyloxycarbonyl, isobomyloxycarbonyl, o-bromobenzyloxycarbonyl, cyclohexyl, isopropyl, acetyl, o- nitrophenylsulfonyl and the like.
  • Typical solid supports are cross-linked polymeric supports. These may include divinylbenzene cross-linked-styrene-based polymers, for example, divinylbenzene-hydroxymethylstyrene copolymers, divinylbenzene-chloromethylstyrene copolymers and divinylbenzene-benzhydrylaminopolystyrene copolymers.
  • divinylbenzene cross-linked-styrene-based polymers for example, divinylbenzene-hydroxymethylstyrene copolymers, divinylbenzene-chloromethylstyrene copolymers and divinylbenzene-benzhydrylaminopolystyrene copolymers.
  • polypeptides of the present disclosure may also be chemically prepared by other methods such as by the method of simultaneous multiple peptide synthesis.
  • the above-described immunogenic polypeptides, polyproteins, and multiepitope fusion proteins may be produced recombinantly.
  • Numerous cloning vectors are known to those of skill in the art, and the selection of an appropriate cloning vector is a matter of choice.
  • a variety of bacterial, yeast, plant, mammalian and insect expression systems are available in the art and any such expression system may be used.
  • a polynucleotide encoding these proteins may be translated in a cell-free translation system. Such methods are well known in the art.
  • Examples of recombinant DNA vectors for cloning and host cells which they may transform include the bacteriophage l (E. coli), pBR322 (E. coli), pACYC177 (E. coli), pKT230 (gram-negative bacteria), pGV1106 (gram-negative bacteria), pLAFRl (gram-negative bacteria), pME290 (non-E. coli gram-negative bacteria), pHV14 (E.
  • coli and Bacillus subtilis pBD9 (Bacillus), pIJ61 (Streptomyces), pUC6 (Streptomyces), YIp5 (Saccharomyces), YCpl9 (Saccharomyces) and bovine papilloma virus (mammalian cells).
  • Insect cell expression systems such as baculovirus systems, may also be used and are known to those of skill in the art and described in, e.g., Summers and Smith, Texas Agricultural Experiment Station Bulletin No. 1555 (1987). Materials and methods for baculovirus/insect cell expression systems are commercially available in kit form from, inter alia, Invitrogen, San Diego, CA ("MaxBac" kit).
  • Plant expression systems may also be used to produce the immunogenic proteins. Generally, such systems use virus-based vectors to transfect plant cells with heterologous genes.
  • Viral systems such as a vaccinia based infection/transfection system, will also find use with the subject matter as disclosed herein.
  • cells are first transfected in vitro with a vaccinia virus recombinant that encodes the bacteriophage T7 RNA polymerase.
  • This polymerase displays extraordinar specificity in that it only transcribes templates bearing T7 promoters.
  • cells are transfected with the DNA of interest, driven by a T7 promoter.
  • the polymerase expressed in the cytoplasm from the vaccinia virus recombinant transcribes the transfected DNA into RNA which is then translated into protein by the host translational machinery.
  • the method provides for high level, transient, cytoplasmic production of large quantities of RNA and its translation product(s).
  • the gene may be placed under the control of a promoter, ribosome binding site (for bacterial expression) and, optionally, an operator (collectively referred to herein as "control" elements), so that the DNA sequence encoding the desired immunogenic polypeptide is transcribed into RNA in the host cell transformed by a vector containing this expression construction.
  • the coding sequence may or may not contain a signal peptide or leader sequence. With the present subject matter as disclosed herein, both the naturally occurring signal peptides or heterologous sequences may be used. Leader sequences may be removed by the host in post- translational processing. See, e.g., U.S. Pat. Nos. 4,431,739; 4,425,437; 4,338,397, each herein incorporated by reference in their entireties. Such sequences include, but are not limited to, the tpa leader, as well as the honey bee mellitin signal sequence.
  • regulatory sequences may also be desirable which allow for regulation of expression of the protein sequences relative to the growth of the host cell.
  • Such regulatory sequences are known to those of skill in the art, and examples include those which cause the expression of a gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • Other types of regulatory elements may also be present in the vector, for example, enhancer sequences.
  • control sequences and other regulatory sequences may be ligated to the coding sequence prior to insertion into a vector.
  • the coding sequence may be cloned directly into an expression vector which already contains the control sequences and an appropriate restriction site.
  • mutants or analogs of the immunogenic polypeptides may be prepared by the deletion of a portion of the sequence encoding the protein, by insertion of a sequence, and/or by substitution of one or more nucleotides within the sequence. Techniques for modifying nucleotide sequences, such as site- directed mutagenesis, are well known to those skilled in the art.
  • the expression vector is then used to transform an appropriate host cell.
  • mammalian cell lines include immortalized cell lines available from the American Type Culture Collection (ATCC), such as, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., Hep G2), as well as others.
  • ATCC American Type Culture Collection
  • CHO Chinese hamster ovary
  • HeLa cells HeLa cells
  • BHK baby hamster kidney
  • COS monkey kidney cells
  • human hepatocellular carcinoma cells e.g., Hep G2
  • bacterial hosts such as E. coli, Bacillus subtilis, and Streptococcus spp., will find use with the present expression constructs.
  • Yeast hosts useful with the subject matter as disclosed include, inter alia, Saccharomyces cerevisiae, Candida albicans, Candida maltosa, Hansenula polymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Pichia guillerimondii, Pichia pastoris, Schizosaccharomyces pombe and Yarrowia lipolytica.
  • Insect cells for use with baculovirus expression vectors include, inter alia, Aedes aegypti, Autographa californica, Bombyx mori, Drosophila melanogaster, Spodoptera frugiperda, and Trichoplusia ni.
  • the proteins as disclosed herein are produced by growing host cells transformed by an expression vector described above under conditions whereby the protein of interest is expressed. The selection of the appropriate growth conditions is within the skill of the art. The cells are then disrupted, using chemical, physical or mechanical means, which lyse the cells yet keep the Sapovirus immunogenic polypeptides substantially intact. Intracellular proteins may also be obtained by removing components from the cell wall or membrane, e.g., by the use of detergents or organic solvents, such that leakage of the immunogenic polypeptides occurs.
  • methods of disrupting cells for use with the subject matter as disclosed herein include but are not limited to: sonication or ultrasonication; agitation; liquid or solid extrusion; heat treatment; freeze-thaw; desiccation; explosive decompression; osmotic shock; treatment with lytic enzymes including proteases such as trypsin, neuraminidase and lysozyme; alkali treatment; and the use of detergents and solvents such as bile salts, sodium dodecyl sulphate, Triton, NP40 and CHAPS.
  • the particular technique used to disrupt the cells is largely a matter of choice and will depend on the cell type in which the polypeptide is expressed, culture conditions and any pre-treatment used.
  • Sapovirus immunogenic polypeptides are further purified, using standard purification techniques such as but not limited to, column chromatography, ion-exchange chromatography, size-exclusion chromatography, electrophoresis, HPLC, immunoabsorbent techniques, affinity chromatography, immunoprecipitation, and the like.
  • one method for obtaining the intracellular Sapovirus immunogenic polypeptides as disclosed herein involves affinity purification, such as by immunoaffmity chromatography using specific antibodies.
  • affinity purification such as by immunoaffmity chromatography using specific antibodies.
  • affinity resin is within the skill in the art.
  • immunogenic polypeptides may be further purified using conventional techniques well known in the art, such as by any of the techniques described above.
  • polypeptides simultaneously (e.g., structural and/or nonstructural proteins from one or more viral strains or viral polypeptides in combination with polypeptide adjuvants). Production of two or more different polypeptides may readily be accomplished by e.g., co-transfecting host cells with constructs encoding the different polypeptides. Co-transfection may be accomplished either in trans or cis, i.e., by using separate vectors or by using a single vector encoding the polypeptides. If a single vector is used, expression of the polypeptides may be driven by a single set of control elements or, alternatively, the sequences coding for the polypeptides may be present on the vector in individual expression cassettes, regulated by individual control elements.
  • the polypeptides described herein may be attached to a solid support.
  • the solid supports which may be used in the practice with the subject matter as disclosed herein include substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
  • substrates such as nitrocellulose (e.g., in membrane or microtiter well form); polyvinylchloride (e.g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.
  • a solid support is first reacted with a solid phase component (e.g., one or more Sapovirus antigens) under suitable binding conditions such that the component is sufficiently immobilized to the support.
  • a solid phase component e.g., one or more Sapovirus antigens
  • immobilization of the antigen to the support may be enhanced by first coupling the antigen to a protein with better binding properties.
  • Suitable coupling proteins include, but are not limited to, macromolecules such as serum albumins including bovine serum albumin (BSA), keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, and other proteins well known to those skilled in the art.
  • BSA bovine serum albumin
  • Other molecules that may be used to bind the antigens to the support include polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and the like. Such molecules and methods of coupling these molecules to the antigens, are well known to those of ordinary skill in the art.
  • polypeptides may be labeled using conventional techniques.
  • Suitable labels include fluorophores, chromophores, radioactive atoms (particularly 32 P and 125 I, electron-dense reagents, enzymes, and ligands having specific binding partners. Enzymes are typically detected by their activity. For example, horseradish peroxidase is usually detected by its ability to convert 3,3',5,5'-tetramethylbenzidine (TMB) to a blue pigment, quantifiable with a spectrophotometer.
  • TMB 3,3',5,5'-tetramethylbenzidine
  • Specific binding partner refers to a protein capable of binding a ligand molecule with high specificity, as for example in the case of an antigen and a monoclonal antibody specific therefor. Other specific binding partners include biotin and avidin or streptavidin, IgG and protein A, and the numerous receptor-ligand couples known in the art. A single label or a combination of labels may be used in the as disclosed herein.
  • compositions as disclosed herein may be administered directly to the subject (e.g., as described above) or, alternatively, delivered ex vivo, to cells derived from the subject, using methods such as those described above.
  • compositions comprising one or more of the immunogenic polypeptides and/or polyproteins multiepitope fusion proteins described herein.
  • Different polypeptides, polyproteins, and multiple epitope fusion proteins may be mixed together in a single formulation.
  • an antigen of the immunogenic composition may be present in more than one polypeptide, or multiple epitope polypeptide, or polyprotein.
  • Sapovirus VP1 comprising immunogenic compositions include lysates from baculovirus expressed insect host cells.
  • said lysates undergo various levels of fractionation or are unfractionated, where the expressed protein is modified or unmodified from its expressed state (e.g., His n tag intact).
  • the immunogenic compositions may comprise a mixture of polypeptides, which in turn may be delivered using the same or different vehicles.
  • Antigens may be administered individually or in combination, in e.g., prophylactic (i.e., to prevent infection) or therapeutic (to treat infection) immunogenic compositions.
  • the immunogenic composition may be given more than once (e.g., a "prime” administration followed by one or more "boosts") to achieve the desired effects.
  • the same composition may be administered in one or more priming and one or more boosting steps. Alternatively, different compositions may be used for priming and boosting.
  • the immunogenic compositions will generally include one or more "pharmaceutically acceptable excipients or vehicles" such as water, saline, glycerol, ethanol, and the like. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • pharmaceutically acceptable excipients or vehicles such as water, saline, glycerol, ethanol, and the like.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, and the like, may be present in such vehicles.
  • Immunogenic compositions will typically, in addition to the components mentioned above, comprise one or more "pharmaceutically acceptable carriers.” These include any carrier which does not itself induce the production of antibodies harmful to the individual receiving the composition. Suitable carriers typically are large, slowly metabolized macromolecules such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Such carriers are well known to those of ordinary skill in the art. A composition may also contain a diluent, such as water, saline, glycerol, and the like.
  • a diluent such as water, saline, glycerol, and the like.
  • auxiliary substance such as a wetting or emulsifying agent, pH buffering substance, and the like, may be present.
  • auxiliary substance such as a wetting or emulsifying agent, pH buffering substance, and the like.
  • compositions as disclosed herein for example, mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates, as well as salts of organic acids such as acetates, proprionates, malonates, or benzoates.
  • mineral salts such as hydrochlorides, hydrobromides, phosphates, or sulfates
  • organic acids such as acetates, proprionates, malonates, or benzoates.
  • Especially useful protein substrates are serum albumins, keyhole limpet hemocyanin, immunoglobulin molecules, thyroglobulin, ovalbumin, tetanus toxoid, and other proteins well known to those of skill in the art.
  • compositions as disclosed may also contain liquids or excipients, such as water, saline, glycerol, dextrose, ethanol, or the like, singly or in combination, as well as substances such as wetting agents, emulsifying agents, or pH buffering agents.
  • Antigens may also be adsorbed to, entrapped within or otherwise associated with liposomes and particulate carriers such as PLG.
  • Antigens may be conjugated to a carrier protein in order to enhance immunogenicity. This is particularly useful in compositions in which a saccharide or carbohydrate antigen is used.
  • Carrier proteins may include, but are not limited to, bacterial toxins or toxoids, such as diphtheria or tetanus toxoids.
  • the CRM197 diphtheria toxoid may be used.
  • Other carrier polypeptides include the N. meningitidis outer membrane protein (EP-A-0372501), synthetic peptides (EP-A-0378881 and EP-A-0427347), heat shock proteins (WO 93/17712 and WO 94/03208), pertussis proteins (WO 98/58668 and EP-A-0471177), protein D from H.
  • a mixture comprises capsular saccharide from both serigraphs A and C, it may be that the ratio (w/w) of MenA saccharide:MenC saccharide is greater than 1 (e.g., 2:1, 3:1, 4:1, 5:1, 10:1 or higher).
  • Different saccharides may be conjugated to the same or different type of carrier protein. Any suitable conjugation reaction may be used, with any suitable linker where necessary.
  • Immunogenic compositions including vaccines as disclosed may be administered in conjunction with other immunoregulatory agents.
  • a vaccine as disclosed herein may include an adjuvant.
  • adjuvants include, but are not limited to, one or more of the following types of adjuvants described below.
  • Mineral containing compositions suitable for use as adjuvants disclosed herein include mineral salts, such as aluminum salts and calcium salts.
  • Salts as disclosed herein includes mineral salts such as hydroxides (e.g., oxyhydroxides), phosphates (e.g., hydroxyphosphates, orthophosphates), sulfates, and the like, or mixtures of different mineral compounds (e.g., a mixture of a phosphate and a hydroxide adjuvant, optionally with an excess of the phosphate), with the compounds taking any suitable form (e.g., gel, crystalline, amorphous, and the like).
  • the mineral containing compositions may also be formulated as a particle of metal salt (WO00/23105).
  • Aluminum salts may be included in vaccines such that the dose of Al 3+ is between 0.2 and 1.0 mg per dose.
  • the aluminum based adjuvant for use as disclosed is alum (aluminum potassium sulfate (A1K(S04)2)), or an alum derivative, such as that formed in-situ by mixing an antigen in phosphate buffer with alum, followed by titration and precipitation with a base such as ammonium hydroxide or sodium hydroxide.
  • Aluminum-based adjuvant for use in vaccine formulations of the present invention is aluminum hydroxide adjuvant (Al(OH)3) or crystalline aluminum oxyhydroxide (AIOOH), which is an excellent adsorbant, having a surface area of approximately 500 m 2 /g.
  • Al(OH)3 aluminum hydroxide adjuvant
  • AIOOH crystalline aluminum oxyhydroxide
  • AIPO4 aluminum phosphate adjuvant
  • aluminum hydroxyphosphate which contains phosphate groups in place of some or all of the hydroxyl groups of aluminum hydroxide adjuvant is provided.
  • aluminum phosphate adjuvants provided herein are amorphous and soluble in acidic, basic and neutral media.
  • the adjuvant as disclosed herein comprises both aluminum phosphate and aluminum hydroxide.
  • the adjuvant has a greater amount of aluminum phosphate than aluminum hydroxide, such as a ratio of 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or greater than 9: 1, by weight aluminum phosphate to aluminum hydroxide.
  • aluminum salts in the vaccine are present at 0.4 to 1.0 mg per vaccine dose, or 0.4 to 0.8 mg per vaccine dose, or 0.5 to 0.7 mg per vaccine dose, or about 0.6 mg per vaccine dose.
  • the aluminum-based adjuvant(s), or ratio of multiple aluminum-based adjuvants, such as aluminum phosphate to aluminum hydroxide is selected by optimization of electrostatic attraction between molecules such that the antigen carries an opposite charge as the adjuvant at the desired pH.
  • adsorbs lysozyme but not albumin at pH 7.4.
  • albumin be the target
  • aluminum hydroxide adjuvant would be selected (i.e., 11.4).
  • pretreatment of aluminum hydroxide with phosphate lowers its isoelectric point, making it a preferred adjuvant for more basic antigens.
  • Oil-emulsion compositions suitable for use as adjuvants may include squalene- water emulsions, such as MF59 (5% Squalene, 0.5% TWEEN 80TM, and 0.5% SPAN 85TM, formulated into submicron particles using a microfluidizer). See WO90/14837. MF59 is used as the adjuvant in the FLU ADTM influenza virus trivalent subunit vaccine.
  • Particularly adjuvants for use in the compositions are submicron oil-in-water emulsions.
  • Submicron oil-in-water emulsions for use herein may be squalene/water emulsions optionally containing varying amounts of MTP-PE, such as a submicron oil-in-water emulsion containing 4-5% w/v squalene, 0.25-1.0% w/v TWEEN 80TM (polyoxyelthylenesorbitan monooleate), and/or 0.25-1.0% SPAN 85TM (sorbitan trioleate), and, optionally, N- acetylmuramyl-L-alanyl-D-isogluatminyl-L-alanine-2-(.beta.-2'-dipalmito- yl-sn-glycero-3- huydroxyphosphosphoryloxy)-ethyl amine (MTP-PE), for example, the submicron
  • MF59 contains 4-5% w/v Squalene (e.g., 4.3%), 0.25-0.5% w/v TWEEN 80TM, and 0.5% w/v SPAN 85TM and optionally contains various amounts of MTP-PE, formulated into submicron particles using a microfluidizer such as Model 110Y microfluidizer (Microfluidics, Newton, Mass.).
  • MTP-PE may be present in an amount of about 0- 500 pg/dose, 0-250 pg/dose and 0-100 pg/dose.
  • MF59-0 refers to the above submicron oil-in-water emulsion lacking MTP-PE
  • MF59-MTP denotes a formulation that contains MTP-PE.
  • MF59-100 contains 100 .mu.g MTP-PE per dose, and so on.
  • MF69 another submicron oil-in-water emulsion for use herein, contains 4.3% w/v squalene, 0.25% w/v TWEEN 80TM, and 0.75% w/v SPAN 85TM and optionally MTP-PE.
  • MF75 also known as SAF, containing 10% squalene, 0.4% TWEEN 80TM, 5% pluronic-blocked polymer L121, and thr-MDP, also microfluidized into a submicron emulsion.
  • MF75-MTP denotes an MF75 formulation that includes MTP, such as from 100-400 pg MTP-PE per dose.
  • Submicron oil-in-water emulsions methods of making the same and immunostimulating agents, such as muramyl peptides, for use in the compositions, are described in detail in International Publication No. WO90/14837 and U.S. Pat. Nos. 6,299,884 and 6,45 1,325.
  • CFA Complete Freund's adjuvant
  • IF A incomplete Freund's adjuvant
  • Saponin formulations may also be used as adjuvants.
  • Saponins are a heterologous group of sterol glycosides and triterpenoid glycosides that are found in the bark, leaves, stems, roots and even flowers of a wide range of plant species. Saponins isolated from the bark of the Quillaia saponaria Molina tree have been widely studied as adjuvants. Saponins may also be commercially obtained from Smilax ornata (sarsaprilla), Gypsophilla paniculata (brides veil), and Saponaria officianalis (soap root).
  • Saponin adjuvant formulations include purified formulations, such as QS21, as well as lipid formulations, such as ISCOMs.
  • Saponin compositions have been purified using High Performance Thin Layer Chromatography (HP-TLC) and Reversed Phase High Performance Liquid Chromatography (RP-HPLC). Specific purified fractions using these techniques have been identified, including QS7, QS17, QS18, QS21, QH-A, QH-B and QH-C.
  • the saponin is QS21.
  • a method of production of QS21 is disclosed in U.S. Pat. No. 5,057,540.
  • Saponin formulations may also comprise a sterol, such as cholesterol (see W096/33739).
  • ISCOMs Immunostimulating Complexes
  • phospholipid such as phosphatidylethanolamine or phosphatidylcholine. Any known saponin may be used in ISCOMs.
  • the ISCOM includes one or more of Quil A, QHA and QHC. ISCOMs are further described in EP0109942, W096/11711 and W096/33739.
  • the ISCOMS may be devoid of (an) additional detergent(s). See WO00/07621.
  • Adjuvants suitable for use as disclosed herein include bacterial or microbial derivatives such as:
  • Non-Toxic Derivatives of Enterobacterial Lipopolysaccharide include Monophosphoryl lipid A (MPL) and 3-O-deacylated MPL (3dMPL).
  • 3dMPL is a mixture of 3 De-O-acylated monophosphoryl lipid A with 4, 5 or 6 acylated chains.
  • One "small particle” form of 3 De-O-acylated monophosphoryl lipid A is disclosed in EP 0 689454.
  • Such "small particles” of 3dMPL are small enough to be sterile filtered through a 0.22 micron membrane (see EP 0689454).
  • Other non-toxic LPS derivatives include monophosphoryl lipid A mimics, such as aminoalkyl glucosaminide phosphate derivatives e.g., RC-529.
  • Lipid A derivatives include derivatives of lipid A from Escherichia coli such as OM- 174.
  • Immunostimulatory oligonucleotides suitable for use as adjuvants may include nucleotide sequences containing a CpG motif (a sequence containing an unmethylated cytosine followed by guanosine and linked by a phosphate bond). Bacterial double stranded RNA or oligonucleotides containing palindromic or poly(dG) sequences have also been shown to be immunostimulatory.
  • the CpG's may include nucleotide modifications/analogs such as phosphorothioate modifications and may be double-stranded or single-stranded.
  • the guanosine may be replaced with an analog such as 2'-deoxy-7-deazaguanosine.
  • the CpG sequence may be directed to TLR9, such as the motif GTCGTT or TTCGTT.
  • the CpG sequence may be specific for inducing a Thl immune response, such as a CpG-A ODN, or it may be more specific for inducing a B cell response, such a CpG-B ODN.
  • the CpG is a CpG-A ODN.
  • the CpG oligonucleotide may be constructed so that the 5' end is accessible for receptor recognition.
  • two CpG oligonucleotide sequences may be attached at their 3' ends to form "immunomers.”
  • [00171] (4) ADP-Ribosylating Toxins and Detoxified Derivatives Thereof.
  • Bacterial ADP-ribosylating toxins and detoxified derivatives thereof may be used as adjuvants.
  • the protein may be derived from E. coli (i.e., E. coli heat labile enterotoxin "LT), cholera ("CT"), or pertussis ("PT").
  • LT E. coli heat labile enterotoxin
  • CT cholera
  • PT pertussis
  • the use of detoxified ADP-ribosylating toxins as mucosal adjuvants is described in W095/17211 and as parenteral adjuvants in W098/42375.
  • the adjuvant is a detoxified LT mutant such as LT-K63, LT-R72, and LTR192G.
  • Bioadhesives and mucoadhesives may also be used as adjuvants.
  • Suitable bioadhesives include esterified hyaluronic acid microspheres or mucoadhesives such as cross-linked derivatives of polyacrylic acid, polyvinyl alcohol, polyvinyl pyrollidone, polysaccharides and carboxymethylcellulose. Chitosan and derivatives thereof may also be used as adjuvants. E.g., WO99/27960.
  • muramyl peptides suitable for use as adjuvants include N-acetyl- muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl-normuramyl-l-alanyl-d-isoglutamine (nor-MDP), and N-acetylmuramyl-l-alanyl-d-isoglutaminyl-l-alanine-2-(r-2'-dipalmitoyl-s- n- glycero-3-hydroxyphosphoryloxy)-ethylamine MTP-PE).
  • thr-MDP N-acetyl- muramyl-L-threonyl-D-isoglutamine
  • nor-MDP N-acetyl-normuramyl-l-alanyl-d-isoglutaminyl-l-alanine-2-(r-2'-dipalmitoyl-s-
  • imidazoquinoline compounds suitable for use as adjuvants include Imiquimod and its analogues (see, e.g., U.S. Pat. Nos. 4,689,338, 5,389,640, 5,268,376, 4,929,624, 5,266,575, 5,352,784, 5,494,916, 5,482,936, 5,346,905, 5,395,937, 5,238,944, and 5,525,612).
  • Thiosemicarbazone Compounds [00176] Examples of thiosemicarbazone compounds, as well as methods of formulating, manufacturing, and screening for compounds all suitable for use as adjuvants include those described in W004/60308. The thiosemicarbazones are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF-a
  • tryptanthrin compounds as well as methods of formulating, manufacturing, and screening for compounds all suitable for use as adjuvants as disclosed herein include those described in WO04/64759.
  • the tryptanthrin compounds are particularly effective in the stimulation of human peripheral blood mononuclear cells for the production of cytokines, such as TNF-a.
  • compositions as disclosed herein may be applied to the compositions as disclosed herein.
  • adjuvant compositions may be used:
  • RIB ITM adjuvant system (RAS), (Ribi Immunochem) containing 2% Squalene, 0.2% TWEEN 80TM, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), MPL+CWS (DETOXTM); and (8) one or more mineral salts (such as an aluminum salt)+a non-toxic derivative of LPS (such as 3dPML).
  • RAS RIB ITM adjuvant system
  • Ribi Immunochem containing 2% Squalene, 0.2% TWEEN 80TM, and one or more bacterial cell wall components from the group consisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM), and cell wall skeleton (CWS), MPL+CWS (DETOXTM); and (8) one or more mineral salts (such as an aluminum salt)+a non-toxic derivative of LPS (such as 3dPML).
  • one or more mineral salts such as an aluminum salt
  • one or more immunostimulatory oligonucleotides such as a nucleotide sequence including a CpG motif
  • one or more detoxified ADP- ribosylating toxins such as LT-K63 and LT-R72.
  • compositions of the as disclosed herein optionally may comprise one or more additional polypeptide antigens which are not derived from Sapovirus proteins.
  • additional polypeptide antigens include bacterial, viral, or parasitic antigens.
  • a Sapovirus antigen is combined with one or more antigens including, but not limited to, antigens derived from a bacteria or virus, such as Orthomyxovirus (influenza), Pneumovirus (RSV), Paramyxovirus (PIV and Mumps), Morbillivirus (measles), Togavirus (Rubella), Enterovirus (polio), HBV, Coronavirus (SARS), and Varicella-zoster virus (VZV), Epstein Barr virus (EBV), Streptococcus pneumoniae, Neisseria meningitides, Streptococcus pyogenes (Group A Streptococcus), Moraxella catarrhalis, Bordetella pertussis, Staphylococcus aureus, Clostridium tetani (Tetanus), Corny neb acterium diphtheriae (Diphtheria), Haemophilus influenzae B (Hib), P
  • a Sapovirus antigen is combined with one or more antigens including, but not limited to, Neisseria meningitides, Streptococcus pneumoniae, Streptococcus pyogenes (Group A Streptococcus), Moraxella catarrhalis, Bordetella pertussis, Staphylococcus aureus, Staphylococcus epidermis, Clostridium tetani (Tetanus), Corny neb acterium diphtheriae (Diphtheria), Haemophilus influenzae B (Hib), Pseudomonas aeruginosa, Legionella pneumophila, Streptococcus agalactiae (Group B Streptococcus), Enterococcus faecalis, Helicobacter pylori, Clamydia pneumoniae, Orthomyxovirus (influenza), Pneumovirus (RSV),
  • a Sapovirus antigen is combined with one or more antigens which are useful in a vaccine designed to protect individuals against pathogens that cause diarrheal diseases.
  • antigens include, but are not limited to, rotavirus, Shigella spp., enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, and Campylobacter jejuni antigens.
  • ETEC enterotoxigenic Escherichia coli
  • Vibrio cholerae Vibrio cholerae
  • Campylobacter jejuni antigens include, but are not limited to, rotavirus, Shigella spp., enterotoxigenic Escherichia coli (ETEC), Vibrio cholerae, and Campylobacter jejuni antigens.
  • one or more Norovirus antigens may be derived from Norwalk virus, Snow Mountain virus, and/or Hawaii virus are combined with a rotavirus antigen in an immunogenic composition.
  • Antigens which may find use with the present compositions include, but are not limited to, one or more of the following antigens set forth below, or antigens derived from one or more of the pathogens set forth below:
  • Suitable Bacterial antigens as disclosed herein include proteins, polysaccharides, lipopolysaccharides, and outer membrane vesicles which may be isolated, purified or derived from a bacteria.
  • bacterial antigens may include bacterial lysates and inactivated bacteria formulations.
  • Bacteria antigens may be produced by recombinant expression.
  • Bacterial antigens include epitopes which may be exposed on the surface of the bacteria during at least one stage of its life cycle. Bacterial antigens may be conserved across multiple serotypes.
  • Bacterial antigens include antigens derived from one or more of the bacteria set forth below as well as the specific antigens examples identified below.
  • Meningitides antigens may include proteins (such as those identified in References 1-7), saccharides (including a polysaccharide, oligosaccharide or lipopolysaccharide), or outer-membrane vesicles purified or derived from N. meningitides serogroup such as A, C, W135, Y, and/or B. Meningitides protein antigens may be selected from adhesions, autotransporters, toxins, Fe acquisition proteins, and membrane associated proteins (e.g., integral outer membrane protein).
  • Streptococcus pneumoniae antigens may include a saccharide (including a polysaccharide or an oligosaccharide) and/or protein from Streptococcus pneumoniae. Saccharide antigens may be selected from serotypes 1, 2, 3, 4, 5, 6B, 7F, 8, 9N, 9V, 10 A, 11 A, 12F, 14, 15B, 17F, 18C, 19 A, 19F, 20, 22F, 23F, and 33F. Protein antigens may be selected from a protein identified in WO 98/18931, WO 98/18930, U.S. Pat. No. 6,699,703, U.S. Pat. No.
  • Streptococcus pneumoniae proteins may be selected from the Poly Histidine Triad family (PhtX), the Choline Binding Protein family (CbpX), CbpX truncates, LytX family, LytX truncates, CbpX truncate-LytX truncate chimeric proteins, pneumolysin (Ply), PspA, PsaA, Spl28, SplOl, Spl30, Spl25 or Spl33.
  • PhtX Poly Histidine Triad family
  • CbpX Choline Binding Protein family
  • CbpX truncates CbpX truncates
  • LytX family LytX truncates
  • pneumolysin (Ply) PspA, PsaA, Spl28, SplOl, Spl30, Spl25 or Spl33.
  • Streptococcus pyogenes Group A Streptococcus antigens may include a protein identified in WO 02/34771 or WO 2005/032582 (including GAS 40), fusions of fragments of GAS M proteins (including those described in WO 02/094851), fibronectin binding protein (Sfbl), Streptococcal heme-associated protein (Shp), and Streptolysin S (SagA).
  • Moraxella catarrhalis Moraxella antigens include antigens identified in WO 02/18595 and WO 99/58562, outer membrane protein antigens (HMW-OMP), C-antigen, and/or LPS.
  • HMW-OMP outer membrane protein antigens
  • C-antigen C-antigen
  • LPS LPS
  • Pertussis antigens include petussis holotoxin (PT) and filamentous haemagglutinin (FHA) from B. pertussis, optionally also combination with pertactin and/or agglutinogens 2 and 3 antigen.
  • PT petussis holotoxin
  • FHA filamentous haemagglutinin
  • Staphylococcus aureus antigens include S. aureus type 5 and 8 capsular polysaccharides optionally conjugated to nontoxic recombinant Pseudomonas aeruginosa exotoxin A, such as STAPHVAXTM, or antigens derived from surface proteins, invasins (leukocidin, kinases, hyaluronidase), surface factors that inhibit phagocytic engulfment (capsule, Protein A), carotenoids, catalase production, Protein A, coagulase, clotting factor, and/or membrane-damaging toxins (optionally detoxified) that lyse eukaryotic cell membranes (hemolysins, leukotoxin, leukocidin).
  • STAPHVAXTM nontoxic recombinant Pseudomonas aeruginosa exotoxin A
  • antigens derived from surface proteins invasins (leukocid
  • Staphylococcus epidermis S. epidermidis antigens include slime-associated antigen (SAA).
  • Tetanus antigens include tetanus toxoid (TT), may be used as a carrier protein in conjunction/conjugated with the compositions of the present disclosure.
  • Diphtheria antigens include diphtheria toxin, including detoxified, such as CRM197. Additionally, antigens capable of modulating, inhibiting or associated with ADP ribosylation are contemplated for combination/co- admini strati on/conjugati on with the compositions of the present disclosure.
  • the diphtheria toxoids may be used as carrier proteins.
  • Hib antigens include a Hib saccharide antigen.
  • Pseudomonas aeruginosa Pseudomonas antigens include endotoxin A, Wzz protein, P. aeruginosa LPS, more particularly LPS isolated from PAOl (05 serotype), and/or Outer Membrane Proteins, including Outer Membrane Proteins F (OprF).
  • Legionella pneumophila Bacterial antigens may be derived from Legionella pneumophila.
  • Streptococcus agalactiae Group B Streptococcus antigens include a protein or saccharide antigen identified in WO 02/34771, WO 03/093306, WO 04/041157, or WO 2005/002619 (including proteins GBS 80, GBS 104, GBS 276 and GBS 322, and including saccharide antigens derived from serotypes la, lb, Ia/c, II, III, IV, V, VI, VII and VIII).
  • Neiserria gonorrhoeae antigens include Por (or porin) protein, such as PorB, a transferring binding protein, such as TbpA and TbpB, a opacity protein (such as Opa), a reduction-modifiable protein (Rmp), and outer membrane vesicle (OMV) preparations (see e.g., W099/24578, W099/36544, WO99/57280, WO02/079243).
  • Chlamydia trachomatis antigens include antigens derived from serotypes A, B, Ba and C (agents of trachoma, a cause of blindness), serotypes Li, L2 & L3 (associated with Lymphogranuloma venereum), and serotypes, D-K.
  • Chlamydia trachomas antigens may also include an antigen identified in WO 00/37494, WO 03/049762, WO 03/068811, or WO 05/002619, including PepA (CT045), LcrE (CT089), ArtJ (CT381), DnaK (CT396), CT398, OmpH-like (CT242), L7/L12 (CT316), OmcA(CT444), AtosS (CT467), CT547, Eno (CT587), HrtA (CT823), and MurG (CT761).
  • Treponema pallidum Syphilis
  • Syphilis antigens include TmpA antigen.
  • Haemophilus ducreyi causing chancroid
  • Ducreyi antigens include outer membrane protein (DsrA).
  • Antigens include a trisaccharide repeat or other Enterococcus derived antigens provided in Ei.S. Pat. No. 6,756,36E
  • H. pylori antigens include Cag, Vac, Nap, HopX, HopY and/or urease antigen.
  • Staphylococcus saprophyticus Antigens include the 160 kDa hemagglutinin of S. saprophyticus antigen.
  • Yersinia enterocolitica Antigens include LPS.
  • E. coli antigens may be derived from enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAggEC), diffusely adhering E. coli (DAEC), enteropathogenic E. coli (EPEC), and/or enterohemorrhagic E. coli (EHEC).
  • ETEC enterotoxigenic E. coli
  • EAggEC enteroaggregative E. coli
  • DAEC diffusely adhering E. coli
  • EPEC enteropathogenic E. coli
  • EHEC enterohemorrhagic E. coli
  • Bacillus anthracis (anthrax): B. anthracis antigens are optionally detoxified and may be selected from A-components (lethal factor (LF) and edema factor (EF)), both of which may share a common B-component known as protective antigen (PA).
  • LF lethal factor
  • EF edema factor
  • PA protective antigen
  • Plague antigens include FI capsular antigen.
  • Tuberculosis antigens include lipoproteins, LPS, BCG antigens, a fusion protein of antigen 85B (Ag85B) and/or ESAT-6 optionally formulated in cationic lipid vesicles, Mycobacterium tuberculosis (Mtb) isocitrate dehydrogenase associated antigens, and/or MPT51 antigens.
  • Antigens include outer membrane proteins, including the outer membrane protein A and/or B (OmpB).
  • Listeria monocytogenes Bacterial antigens may be derived from Listeria monocytogenes.
  • Chlamydia pneumoniae Antigens include those identified in WO 02/02606.
  • Vibrio cholerae Antigens include proteinase antigens, LPS, particularly lipopoly saccharides of Vibrio cholerae II, 01 Inaba O-specific polysaccharides, V. cholera 0139, antigens of IEM108 vaccine, and/or Zonula occludens toxin (Zot).
  • Salmonella typhi typhoid fever
  • Antigens include capsular polysaccharides, including conjugates (Vi, i.e., vax-TyVi).
  • Antigens include lipoproteins (such as OspA, OspB, Osp C and Osp D), other surface proteins such as OspE-related proteins (Erps), decorin binding proteins (such as DbpA), and antigenically variable VI proteins., such as antigens associated with P39 and P13 VlsE Antigenic Variation Protein.
  • Porphyromonas gingivalis Antigens include P. gingivalis outer membrane protein (OMP).
  • OMP outer membrane protein
  • Klebsiella Antigens include an OMP, including OMP A, or a polysaccharide optionally conjugated to tetanus toxoid.
  • Further bacterial antigens of the instant disclosure may be capsular antigens, polysaccharide antigens or protein antigens of any of the above. Further bacterial antigens may also include an outer membrane vesicle (OMV) preparation. Additionally, antigens include live, attenuated, and/or purified versions of any of the aforementioned bacteria.
  • the antigens of the present disclosure may be derived from gram-negative or gram-positive bacteria.
  • the antigens of the present disclosure may be derived from aerobic or anaerobic bacteria.
  • any of the above bacterial-derived saccharides may be conjugated to another agent or antigen, such as a carrier protein (for example CRM197).
  • a carrier protein for example CRM197
  • conjugation may be direct conjugation effected by reductive amination of carbonyl moieties on the saccharide to amino groups on the protein, as provided in U.S. Pat. No. 5,360,897.
  • the saccharides may be conjugated through a linker, such as, with succinamide or other linkages.
  • Viral antigens suitable for use in the compositions as disclosed include purified subunit formulations, viral proteins which may be isolated, purified or derived from a virus, and Virus Like Particles (VLPs). Viral antigens may be derived from viruses propagated on cell culture or other substrate. Alternatively, viral antigens may be expressed recombinantly. Viral antigens include epitopes which are exposed on the surface of the virus during at least one stage of its life cycle. Viral antigens may be conserved across multiple serotypes or isolates. Viral antigens include antigens derived from one or more of the viruses set forth below as well as the specific antigens examples identified below.
  • Orthomyxovirus Viral antigens may be derived from an Orthomyxovirus, such as Influenza A, B and C. Orthomyxovirus antigens may be selected from one or more of the viral proteins, including hemagglutinin (HA), neuraminidase (NA), nucleoprotein (NP), matrix protein (Ml), membrane protein (M2), one or more of the transcriptase components (PB1, PB2 and PA). In embodiments, antigens include HA and NA.
  • HA hemagglutinin
  • NA neuraminidase
  • NP nucleoprotein
  • Ml matrix protein
  • M2 membrane protein
  • antigens include HA and NA.
  • Influenza antigens may be derived from interpandemic (annual) flu strains.
  • influenza antigens may be derived from strains with the potential to cause pandemic a pandemic outbreak (i.e., influenza strains with new haemagglutinin compared to the haemagglutinin in currently circulating strains, or influenza strains which are pathogenic in avian subjects and have the potential to be transmitted horizontally in the human population, or influenza strains which are pathogenic to humans).
  • Paramyxoviridae viruses Viral antigens may be derived from Paramyxoviridae viruses, such as Pneumoviruses (RSV), Paramyxoviruses (PIV) and Morbilliviruses (Measles).
  • RSV Pneumoviruses
  • PMV Paramyxoviruses
  • Measles Morbilliviruses
  • Pneumovirus Viral antigens may be derived from a Pneumovirus, such as Respiratory syncytial virus (RSV), Bovine respiratory syncytial virus, Pneumonia virus of mice, and Turkey rhinotracheitis virus.
  • the Pneumovirus is RSV.
  • Pneumovirus antigens may be selected from one or more of the following proteins, including surface proteins Fusion (F), Glycoprotein (G) and Small Hydrophobic protein (SH), matrix proteins M and M2, nucleocapsid proteins N, P and L and nonstructural proteins NS1 and NS2.
  • Pneumovirus antigens may include F, G and M.
  • Pneumovirus antigens may also be formulated in or derived from chimeric viruses.
  • chimeric RSV/PIV viruses may comprise components of both RSV and PIV.
  • Paramyxovirus Viral antigens may be derived from a Paramyxovirus, such as Parainfluenza virus types 1-4 (PIV), Mumps, Sendai viruses, Simian virus 5, Bovine parainfluenza virus and Newcastle disease virus.
  • the Paramyxovirus is PIV or Mumps.
  • Paramyxovirus antigens may be selected from one or more of the following proteins: Hemagglutinin-Neuraminidase (HN), Fusion proteins FI and F2, Nucleoprotein (NP), Phosphoprotein (P), Large protein (L), and Matrix protein (M).
  • Paramyxovirus proteins may include HN, FI and F2.
  • Paramyxovirus antigens may also be formulated in or derived from chimeric viruses.
  • chimeric RSV/PIV viruses may comprise components of both RSV and PIV.
  • Commercially available mumps vaccines include live attenuated mumps virus, in either a monovalent form or in combination with measles and rubella vaccines (MMR).
  • Morbillivirus Viral antigens may be derived from a Morbillivirus, such as Measles. Morbillivirus antigens may be selected from one or more of the following proteins: hemagglutinin (H), Glycoprotein (G), Fusion factor (F), Large protein (L), Nucleoprotein (NP), Polymerase phosphoprotein (P), and Matrix (M).
  • H hemagglutinin
  • G Glycoprotein
  • F Fusion factor
  • L Large protein
  • NP Nucleoprotein
  • P Polymerase phosphoprotein
  • M Matrix
  • Commercially available measles vaccines include live attenuated measles virus, typically in combination with mumps and rubella (MMR).
  • Picornavirus Viral antigens may be derived from Picornaviruses, such as Enteroviruses, Rhinoviruses, Hepamavirus, Cardioviruses and Aphthoviruses. Antigens derived from Enteroviruses, such as Poliovirus are may be used.
  • Enterovirus Viral antigens may be derived from an Enterovirus, such as Poliovirus types 1, 2 or 3, Coxsackie A virus types 1 to 22 and 24, Coxsackie B virus types 1 to 6, Echovirus (ECHO) virus) types 1 to 9, 11 to 27 and 29 to 34 and Enterovirus 68 to 71.
  • the Enterovirus may be poliovirus.
  • Enterovirus antigens may include one or more of the following Capsid proteins VP1, VP2, VP3 and VP4.
  • Commercially available polio vaccines include Inactivated Polio Vaccine (IPV) and Oral poliovirus vaccine (OPV).
  • Hepamavirus Viral antigens may be derived from an Hepamavirus, such as Hepatitis A vims (HAV). Commercially available HAV vaccines include inactivated HAV vaccine.
  • Togavirus Viral antigens may be derived from a Togavirus, such as a Rubivirus, an Alphavirus, or an Arterivirus. Antigens derived from Rubivirus, such as Rubella virus, may be used. Togavirus antigens may be selected from El, E2, E3, C, NSP-1, NSPO-2, NSP-3 or NSP-4. Togavirus antigens include El, E2 or E3.
  • Commercially available Rubella vaccines include a live cold-adapted virus, typically in combination with mumps and measles vaccines (MMR).
  • Flavivirus Viral antigens may be derived from a Flavivirus, such as Tick-borne encephalitis (TBE), Dengue (types 1, 2, 3 or 4), Yellow Fever, Japanese encephalitis, West Nile encephalitis, St. Louis encephalitis, Russian spring-summer encephalitis, Powassan encephalitis. Flavivirus antigens may be selected from PrM, M, C, E, NS-1, NS-2a, NS2b, NS3, NS4a, NS4b, and NS5. Flavivirus antigens may include PrM, M and E. Commercially available TBE vaccine include inactivated virus vaccines.
  • Pestivirus Viral antigens may be derived from a Pestivirus, such as Bovine viral diarrhea (BVDV), Classical swine fever (CSFV) or Border disease (BDV).
  • BVDV Bovine viral diarrhea
  • CSFV Classical swine fever
  • BDV Border disease
  • Hepadnavirus Viral antigens may be derived from a Hepadnavirus, such as Hepatitis B virus. Hepadnavirus antigens may be selected from surface antigens (L, M and S), core antigens (HBc, HBe). Commercially available HBV vaccines include subunit vaccines comprising the surface antigen S protein.
  • Hepatitis C virus Viral antigens may be derived from a Hepatitis C virus (HCV). HCV antigens may be selected from one or more of El, E2, E1/E2, NS345 polyprotein, NS 345-core polyprotein, core, and/or peptides from the nonstructural regions.
  • Rhabdovirus Viral antigens may be derived from a Rhabdovirus, such as a Lyssavirus (Rabies virus) and Vesiculovirus (VSV). Rhabdovirus antigens may be selected from glycoprotein (G), nucleoprotein (N), large protein (L), nonstructural proteins (NS).
  • Rhabdovirus antigens may be selected from glycoprotein (G), nucleoprotein (N), large protein (L), nonstructural proteins (NS).
  • G glycoprotein
  • N nucleoprotein
  • L large protein
  • NS nonstructural proteins
  • Rabies virus vaccine comprise killed virus grown on human diploid cells or fetal rhesus lung cells.
  • Viral antigens may be derived from Calciviridae, such as Norwalk virus, and Norwalk-like Viruses, such as Hawaii Virus and Snow Mountain Virus.
  • Coronavirus Viral antigens may be derived from a Coronavirus, SARS, Human respiratory coronavirus, Avian infectious bronchitis (IBV), Mouse hepatitis virus (MHV), and Porcine transmissible gastroenteritis virus (TGEV). Coronavirus antigens may be selected from spike (S), envelope (E), matrix (M), nucleocapsid (N), and Hemagglutinin-esterase glycoprotein (HE).
  • the Coronavirus antigen is derived from a SARS virus.
  • SARS viral antigens are described in WO 04/92360;
  • Retrovirus Viral antigens may be derived from a Retrovirus, such as an Oncovirus, a Lentivirus or a Spumavirus.
  • Oncovirus antigens may be derived from HTLV-1, HTLV-2 or HTLV-5.
  • Lentivirus antigens may be derived from HIV-1 or HIV-2.
  • Retrovirus antigens may be selected from gag, pol, env, tax, tat, rex, rev, nef, vif, vpu, and vpr.
  • HIV antigens may be selected from gag (p24gag and p55gag), env (gpl60 and gp41), pol, tat, nef, rev vpu, miniproteins, (e.g., p55 gag and gpl40v delete). HIV antigens may be derived from one or more of the following strains: HIVm b , HIV S F2, HIVLAV, HIVLAI, HIVMN, HIV-1 C M235, HIV-1US4.
  • Reovirus Viral antigens may be derived from a Reovirus, such as an Orthoreovirus, a Rotavirus, an Orbivirus, or a Coltivirus.
  • Reovirus antigens may be selected from structural proteins l ⁇ , l 2, l3, m ⁇ , m2, s ⁇ , s2, or s3, or nonstructural proteins oNS, pNS, or s 1 s.
  • Reovirus antigens may be derived from a Rotavirus.
  • Rotavirus antigens may be selected from VP1, VP2, VP3, VP4 (or the cleaved product VP5 and VP8), NSP 1, VP6, NSP3, NSP2, VP7, NSP4, or NSP5.
  • Rotavirus antigens may include VP4 (or the cleaved product VP5 and VP8), and VP7.
  • Parvovirus Viral antigens may be derived from a Parvovirus, such as Parvovirus B19. Parvovirus antigens may be selected from VP-1, VP-2, VP-3, NS-1 and NS-2. In embodiments, the Parvovirus antigen is capsid protein VP-2.
  • HDV Delta hepatitis virus
  • Viral antigens may be derived HDV, particularly .delta.- antigen from HDV (see, e.g., U.S. Pat. No. 5,378,814).
  • Hepatitis E virus Viral antigens may be derived from HEV.
  • HEV Hepatitis G virus
  • HGV Viral antigens may be derived from HGV.
  • Human Herpesvirus Viral antigens may be derived from a Human Herpesvirus, such as Herpes Simplex Viruses (HSV), Varicella-zoster virus (VZV), Epstein-Barr virus (EBV), Cytomegalovirus (CMV), Human Herpesvirus 6 (HHV6), Human Herpesvirus 7 (HHV7), and Human Herpesvirus 8 (HHV8).
  • Human Herpesvirus antigens may be selected from immediate early proteins (a), early proteins (b), and late proteins (g).
  • HSV antigens may be derived from HSV-1 or HSV-2 strains.
  • HSV antigens may be selected from glycoproteins gB, gC, gD and gH, fusion protein (gB), or immune escape proteins (gC, gE, or gl).
  • VZV antigens may be selected from core, nucleocapsid, tegument, or envelope proteins. Alive attenuated VZV vaccine is commercially available.
  • EBV antigens may be selected from early antigen (EA) proteins, viral capsid antigen (VCA), and glycoproteins of the membrane antigen (MA).
  • CMV antigens may be selected from capsid proteins, envelope glycoproteins (such as gB and gH), and tegument proteins
  • Papovaviruses Antigens may be derived from Papovaviruses, such as Papillomaviruses and Polyomaviruses.
  • Papillomaviruses include HPV serotypes 1, 2, 4, 5, 6, 8, 11, 13, 16, 18, 31, 33, 35, 39, 41, 42, 47, 51, 57, 58, 63 and 65.
  • HPV antigens are derived from serotypes 6, 11, 16 or 18.
  • HPV antigens may include capsid proteins (LI) and (L2), or E1-E7, or fusions thereof.
  • Polyomyavirus viruses include BK virus and JK virus.
  • Polyomavirus antigens may be selected from VPl, VP2 or VP3.
  • Circovirus Antigens may be derived from Circoviruses, such as Porcine circovirus (PCV) 1, PCV 2, PCV 3, and PCV 4.
  • PCV Porcine circovirus
  • Suitable fungal antigens may be derived from one or more of the fungi set forth below.
  • Fungal antigens may be derived from Dermatophytres, including: Epidermophyton floccusum, Microsporum audouini, Microsporum canis, Microsporum distortum, Microsporum equinum, Microsporum gypsum, Microsporum nanum, Trichophyton concentricum, Trichophyton equinum, Trichophyton gallinae, Trichophyton gypseum, Trichophyton megnini, Trichophyton mentagrophytes, Trichophyton quinckeanum, Trichophyton rubrum, Trichophyton schoenleini, Trichophyton tonsurans, Trichophyton verrucosum, T. verrucosum var. album, var. discoides, var. ochraceum, Trichophyton violaceum, and/or Trichophyton faviforme.
  • Dermatophytres including: Epidermophyton floccusum, Microsporum audouini,
  • Fungal pathogens may be derived from Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, Aspergillus terreus, Aspergillus sydowi, Aspergillus flavatus, Aspergillus glaucus, Blastoschizomyces capitatus, Candida albicans, Candida enolase, Candida tropicalis, Candida glabrata, Candida krusei, Candida parapsilosis, Candida stellatoidea, Candida kusei, Candida parakwsei, Candida lusitaniae, Candida pseudotropicalis, Candida guilliermondi, Cladosporium carrionii, Coccidioides immitis, Blastomyces dermatidis, Cryptococcus neoformans, Geotrichum clavatum, Histoplasma capsulatum, Klebsiella pneumoniae, Paracoccidioides
  • a solubilized fraction extracted and separated from an insoluble fraction obtainable from fungal cells of which cell wall has been substantially removed or at least partially removed characterized in that the process comprises the steps of: obtaining living fungal cells; obtaining fungal cells of which cell wall has been substantially removed or at least partially removed; bursting the fungal cells of which cell wall has been substantially removed or at least partially removed; obtaining an insoluble fraction; and extracting and separating a solubilized fraction from the insoluble fraction.
  • compositions of the as disclosed herein may include one or more antigens derived from a pathogen which causes respiratory disease.
  • respiratory antigens may be derived from a respiratory virus such as Orthomyxoviruses (influenza), Pneumovirus (RSV), Paramyxovirus (PIV), Morbillivirus (measles), Togavirus (Rubella), VZV, and Coronavirus (SARS).
  • Respiratory antigens may be derived from a bacteria which causes respiratory disease, such as Streptococcus pneumoniae, Pseudomonas aeruginosa, Bordetella pertussis, Mycobacterium tuberculosis, Mycoplasma pneumoniae, Chlamydia pneumoniae, Bacillus anthracis, and Moraxella catarrhalis. Examples of specific antigens derived from these pathogens are described above.
  • the immunogenic compositions as disclosed herein may be prepared in various forms.
  • the compositions may be prepared as injectables, either as liquid solutions or suspensions.
  • Solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection may also be prepared (e.g., a lyophilized composition or a spray-freeze dried composition).
  • the composition may be prepared for topical administration e.g., as an ointment, cream or powder.
  • the composition may be prepared for oral administration e.g., as a tablet or capsule or as a spray.
  • the composition may be prepared for pulmonary administration e.g., as an inhaler, using a fine powder or a spray.
  • the composition may be prepared as a suppository or pessary.
  • composition may be prepared for nasal, aural or ocular administration e.g., as drops. Preparation of such pharmaceutical compositions is within the general skill of the art. See, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 18th edition, 1990.
  • the composition may be in kit form, designed such that a combined composition is reconstituted just prior to administration to a patient.
  • kits may comprise one or more Sapovirus antigens or nucleic acids encoding such antigens in liquid form, and any of the additional antigens and adjuvants as described herein.
  • Immunogenic compositions comprising polypeptide antigens as disclosed are vaccine compositions.
  • the pH of such compositions is between 6 and 8, about 7.
  • the pH may be maintained by the use of a buffer.
  • the composition may be sterile and/or pyrogen-free.
  • the composition may be isotonic with respect to the subject.
  • Vaccines according to the instant disclosure may be used either prophylactically or therapeutically, but will typically be prophylactic and may be used to treat animals (including farm, game, companion and laboratory mammals).
  • Immunogenic compositions used as vaccines comprise an immunologically effective amount of antigen(s) and/or nucleic acids encoding antigen(s), as well as any other components, as needed.
  • immunologically effective amount it is meant that the administration of that amount to an individual, either in a single dose or as part of a series, is effective for treatment or prevention. This amount varies depending upon the health and physical condition of the individual to be treated, age, the taxonomic group of individual to be treated (e.g., swine, cattle, and the like), the capacity of the individual's immune system to synthesize antibodies, the degree of protection desired, the formulation of the vaccine, the treating veterinarian's assessment of the medical situation, and other relevant factors. It is expected that the amount will fall in a relatively broad range that may be determined through routine trials.
  • compositions of as disclosed herein will generally be administered directly to a subject.
  • Direct delivery may be accomplished by parenteral injection (e.g., subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue), or mucosally, such as by rectal, oral (e.g., tablet, spray), vaginal, topical, transdermal (see, e.g., W099/27961) or transcutaneous (see e.g., WO02/074244 and W002/064162), intranasal (see, e.g., W003/028760), ocular, aural, pulmonary or other mucosal administration.
  • parenteral injection e.g., subcutaneously, intraperitoneally, intravenously, intramuscularly, or to the interstitial space of a tissue
  • mucosally such as by rectal, oral (e.g., tablet, spray), vaginal, topical, transdermal (
  • Immunogenic compositions may also be administered topically by direct transfer to the surface of the skin. Topical administration may be accomplished without utilizing any devices, or by contacting naked skin with the immunogenic composition utilizing a bandage or a bandage-like device (see, e.g., U.S. Pat. No. 6,348,450).
  • the mode of administration may be parenteral, mucosal or a combination of mucosal and parenteral immunizations.
  • the mode of administration is parenteral, mucosal or a combination of mucosal and parenteral immunizations in a total of 1-2 vaccinations 1-3 weeks apart.
  • the route of administration includes but is not limited to oral delivery, intra-muscular delivery and a combination of oral and intra-muscular delivery.
  • the method for treating an infection by a Sapovirus comprises mucosally administering to a subject in need thereof a first immunogenic composition comprising one or more Sapovirus antigens followed by parenterally administering a therapeutically effective amount of a second immunogenic composition comprising one or more Sapovirus antigens.
  • the immunogenic composition may be used to elicit systemic and/or mucosal immunity, to elicit an enhanced systemic and/or mucosal immunity.
  • the immune response is characterized by the induction of a serum IgG and/or intestinal IgA immune response.
  • prime-boost methods may be employed where one or more gene delivery vectors and/or polypeptide antigens are delivered in a "priming” step and, subsequently, one or more second gene delivery vectors and/or polypeptide antigens are delivered in a "boosting" step.
  • priming and boosting with one or more gene delivery vectors or polypeptide antigens described herein is followed by additional boosting with one or more polypeptide-containing compositions (e.g., polypeptides comprising Sapovirus antigens).
  • the various compositions may be delivered in any order.
  • the nucleic acids need not be all delivered before the polypeptides.
  • the priming step may include delivery of one or more polypeptides and the boosting comprises delivery of one or more nucleic acids and/or one or more polypeptides.
  • Multiple polypeptide administrations may be followed by multiple nucleic acid administrations or polypeptide and nucleic acid administrations may be performed in any order.
  • one or more of the gene delivery vectors described herein and one or more of the polypeptides described herein may be co-administered in any order and via any administration route. Therefore, any combination of polynucleotides and polypeptides described herein may be used to elicit an immune reaction.
  • Dosage treatment may be according to a single dose schedule or a multiple dose schedule. Multiple doses may be used in a primary immunization schedule and/or in a booster immunization schedule. In a multiple dose schedule, the various doses may be given by the same or different routes, e.g., a parenteral prime and mucosal boost, a mucosal prime and parenteral boost, and the like.
  • the dosage regime enhances the avidity of the antibody response leading to antibodies with a neutralizing characteristic.
  • An in-vitro neutralization assay may be used to test for neutralizing antibodies.
  • One way of assessing efficacy of therapeutic treatment involves monitoring infection after administration of a composition of the as disclosed.
  • One way of assessing efficacy of prophylactic treatment involves monitoring immune responses against the antigens in the compositions of the as disclosed after administration of the composition.
  • Another way of assessing the immunogenicity of the component proteins of the immunogenic compositions of the present disclosure is to express the proteins recombinantly and to screen patient sera or mucosal secretions by immunoblot. A positive reaction between the protein and the patient serum indicates that the patient has previously mounted an immune response to the protein in question—that is, the protein is an immunogen. This method may also be used to identify immunodominant proteins and/or epitopes.
  • Another way of checking efficacy of therapeutic treatment involves monitoring infection after administration of the compositions of the present disclosure.
  • One way of checking efficacy of prophylactic treatment involves monitoring immune responses both systemically (such as monitoring the level of IgGl and IgG2a production) and mucosally (such as monitoring the level of IgA production) against the antigens in the compositions of the present disclosure after administration of the composition.
  • immune responses both systemically (such as monitoring the level of IgGl and IgG2a production) and mucosally (such as monitoring the level of IgA production) against the antigens in the compositions of the present disclosure after administration of the composition.
  • serum specific antibody responses are determined post-immunization but pre-challenge whereas mucosal specific antibody body responses are determined post-immunization and post-challenge.
  • the immunogenic compositions of the present disclosure may be evaluated in in vitro and in vivo animal models prior to host.
  • Particularly useful mouse models include those in which intraperitoneal immunization is followed by either intraperitoneal challenge or intranasal challenge.
  • the efficacy of immunogenic compositions of the present disclosure may also be determined in vivo by challenging animal models of infection, e.g., guinea pigs or mice or rhesus macaques, with the immunogenic compositions.
  • the immunogenic compositions may or may not be derived from the same strains as the challenge strains.
  • the immunogenic compositions may be derivable from the same strains as the challenge strains.
  • In vivo efficacy models include but are not limited to: (i) A murine infection model using human strains; (ii) a murine disease model which is a murine model using a mouse-adapted strain, such as strains which are particularly virulent in mice and (iii) a primate model using human isolates.
  • a human challenge model which is supported by the NIH and Center for Disease Control (CDC) is also available.
  • the immune response may be one or both of a THl immune response and a TH2 response.
  • the immune response may be an improved or an enhanced or an altered immune response.
  • the immune response may be one or both of a systemic and a mucosal immune response. In embodiments, the immune response is an enhanced systemic and/or mucosal response.
  • an enhanced systemic and/or mucosal immunity is reflected in an enhanced THl and/or TH2 immune response.
  • the enhanced immune response includes an increase in the production of IgGl and/or IgG2a and/or IgA.
  • the mucosal immune response is a TH2 immune response.
  • the mucosal immune response includes an increase in the production of IgA.
  • Activated TH2 cells enhance antibody production and are therefore of value in responding to extracellular infections.
  • Activated TH2 cells may secrete one or more of IL-4, IL-5, IL-6, and IL-10.
  • a TH2 immune response may result in the production of IgGl, IgE, IgA and memory B cells for future protection.
  • a TH2 immune response may include one or more of an increase in one or more of the cytokines associated with a TH2 immune response (such as IL-4, IL-5, IL-6 and IL-10), or an increase in the production of IgGl, IgE, IgA and memory B cells.
  • the enhanced TH2 immune response will include an increase in IgGl production.
  • ATH1 immune response may include one or more of an increase in CTLs, an increase in one or more of the cytokines associated with a TH 1 immune response (such as IL-2, IFNy, and TNFP), an increase in activated macrophages, an increase in NK activity, or an increase in the production of IgG2a.
  • the enhanced TH1 immune response will include an increase in IgG2a production.
  • Immunogenic compositions of the present disclosure in particular, immunogenic composition comprising one or more antigens of the present disclosure may be used either alone or in combination with other antigens optionally with an immunoregulatory agent capable of eliciting a Thl and/or Th2 response.
  • the immunogenic composition of the present disclosure may also comprise one or more immunoregulatory agents, such as a mineral salt, such as an aluminum salt and an oligonucleotide containing a CpG motif.
  • the immunogenic composition includes both an aluminum salt and an oligonucleotide containing a CpG motif.
  • the immunogenic composition includes an ADP ribosylating toxin, such as a detoxified ADP ribosylating toxin and an oligonucleotide containing a CpG motif.
  • the one or more immunoregulatory agents include an adjuvant.
  • the adjuvant may be selected from one or more of the group consisting of a THl adjuvant and TH2 adjuvant, further discussed above.
  • the immunogenic compositions of the present composition may elicit both a cell mediated immune response as well as a humoral immune response in order to effectively address an infection. This immune response may induce long lasting (e.g., neutralizing) antibodies and a cell mediated immunity that may quickly respond upon exposure to one or more infectious antigens.
  • neutralizing antibodies evidence of neutralizing antibodies in a subject's blood samples is considered as a surrogate parameter for protection since their formation is of decisive importance for virus elimination in TBE infections.
  • the instant disclosure also provides a composition for use as a medicament.
  • the medicament may be able to raise an immune response in a mammal (i.e., it is an immunogenic composition) and may be a vaccine.
  • the present disclosure also provides the use of the instant compositions in the manufacture of a medicament for raising an immune response in a mammal.
  • the medicament may be a vaccine.
  • the vaccine is used to prevent and/or treat an intestinal infection such as gastroenteritis, including acute gastroenteritis.
  • the gastroenteritis may result from an imbalance in ion and/or water transfer resulting in both watery diarrhea and/or intestinal peri stall sis and/or motility (vomiting).
  • the instant disclosure provides methods for inducing or increasing an immune response using the compositions described above.
  • the immune response may be protective and may induce antibodies and/or cell-mediated immunity (including systemic and mucosal immunity). Immune responses include booster responses.
  • the present disclosure also provides a method for raising an immune response in a mammal comprising the step of administering an effective amount of a composition of the instant disclosure.
  • the immune response may be protective and may involve antibodies and/or cell- mediated immunity.
  • the immune response includes one or both of a THl immune response and a TH2 immune response. The method may raise a booster response.
  • Kits comprising one or more containers of compositions as described herein.
  • Compositions may be in liquid form or may be lyophilized, as may individual antigens.
  • Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes.
  • Containers may be formed from a variety of materials, including glass or plastic.
  • a container may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle).
  • the kit may further comprise a second container comprising a pharmaceutically- acceptable buffer, such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery device.
  • a pharmaceutically- acceptable buffer such as phosphate-buffered saline, Ringer's solution, or dextrose solution. It may also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery device.
  • the kit may further include a third component comprising an adjuvant.
  • the kit may also comprise a package insert containing written instructions for methods of inducing immunity or for treating infections.
  • the package insert may be an unapproved draft package insert or may be a package insert approved by the Food and Drug Administration (FDA) or other regulatory body.
  • FDA Food and Drug Administration
  • a delivery device is pre-filled with the immunogenic compositions as disclosed herein.
  • the Sapovirus polypeptides described herein may be used to produce Sapovirus- specific polyclonal and monoclonal antibodies that specifically bind to/are selective for Sapovirus antigens, respectively.
  • Polyclonal antibodies may be produced by administering a Sapovirus polypeptide to a mammal, such as a mouse, a rabbit, a goat, or a horse. Serum from the immunized animal is collected and the antibodies are purified from the plasma by, for example, precipitation with ammonium sulfate, followed by chromatography, including affinity chromatography. Techniques for producing and processing polyclonal antisera are known in the art.
  • Monoclonal antibodies directed against Sapovirus-specific epitopes present in the polypeptides may also be readily produced.
  • Normal B cells from a mammal, such as a mouse, immunized with a Sapovirus polypeptide may be fused with, for example, HAT-sensitive mouse myeloma cells to produce hybridomas.
  • Hybridomas producing Sapovirus-specific antibodies may be identified using RIA or ELISA and isolated by cloning in semi-solid agar or by limiting dilution. Clones producing Sapovirus-specific antibodies are isolated by another round of screening.
  • Antibodies i.e., monoclonal and antibodies from polyclonal sera (polyclonal), which are directed against Sapovirus epitopes, are particularly useful for detecting the presence of Sapovirus antigens in a sample, such as a serum sample from a Sapovirus-infected human.
  • An immunoassay for a Sapovirus antigen may utilize one antibody or several antibodies.
  • An immunoassay for a Sapovirus antigen may use, for example, a monoclonal antibody directed towards a Sapovirus epitope, a combination of monoclonal antibodies directed towards epitopes of one Sapovirus polypeptide, monoclonal antibodies directed towards epitopes of different Sapovirus polypeptides, polyclonal antibodies directed towards the same Sapovirus antigen, polyclonal antibodies directed towards different Sapovirus antigens, or a combination of monoclonal and polyclonal antibodies.
  • Immunoassay protocols may be based, for example, upon competition, direct reaction, or sandwich type assays using, for example, labeled antibody.
  • the labels may be, for example, fluorescent, chemiluminescent, or radioactive.
  • the polyclonal or monoclonal antibodies may further be used to isolate Sapovirus particles or antigens by immunoaffmity columns.
  • the antibodies may be affixed to a solid support by, for example, adsorption or by covalent linkage so that the antibodies retain their immunoselective activity.
  • spacer groups may be included so that the antigen binding site of the antibody remains accessible.
  • the immobilized antibodies may then be used to bind Sapovirus particles or antigens from a biological sample, such as blood or plasma.
  • the bound Sapovirus particles or antigens are recovered from the column matrix by, for example, a change in pH.
  • the antigens recovered may be sequenced, and the sequences used to make constructs for recombinant expression of the recovered antigen by molecular biological means.
  • construct means an artificially-designed segment of nucleic acid (DNA or RNA) borne on a vector that can be used to incorporate genetic material into a target tissue or cell.
  • mice 40 female Balb/c mice were randomly placed into 10 cages of 4 mice each. Mice were allowed to acclimate. Seven cages of mice (total of 28 mice) were vaccinated subcutaneously with a 0.2 ml dose containing formulated with VRx 19030 (recombinant sapovirus protein; SEQ ID NO:2), serial number PV1070820 after mixing with Emulsigen serial PV0080621. The remaining 3 cages of mice (total of 12 mice) were held as non-vaccinated controls. Vaccinated mice were boostered at Day 21 with the same serials and exsanguinated on Day 35. Whole blood was separated into sera. Sera was kept as individual samples as well as pools generated proportionally for the samples from each of 4 mice contained in a specific cage. These pools were treated as 1 individual sample.
  • VRx 19030 recombinant sapovirus protein
  • Sera was tested via indirect ELISA in duplicate, where plates were coated with 0.1 pg V19030 (recombinant Sapovirus protein) or V19045 (recombinant IAV-S H3 protein) diluted in carbonate coating buffer. Plates were washed 3x with lx PBS-T. Antigen plates were then blocked with 1% polyvinyl alcohol diluted in PBS for 1 hour at 37°C. Block was removed. Five fold serial dilutions starting at 1 : 1000 were performed of the sera in duplicate. Sera was allowed to incubate for 1 hour at 37°C. Plates were washed 3x with lx PBS-T.
  • Goat-anti-mouse IgG (H+L) horseradish peroxidase (HRP) conjugate was diluted 1:10000 (Table 1) or 1:17.5000 (Table 2), added to plates and allowed to incubate for 1 hour at 37°C. Plates were washed 3x with lx PBS-T. TMB (3,3',5,5'-Tetramethylbenzidine) substrate was added to plates and allowed to incubate at RT (room temperature) for 3 minutes (Table 1) or 6.5 minutes (Table 2). Plates were stopped with IN H 2 SO 4 (sulfuric acid) and read for OD at 450 nm.
  • HRP horseradish peroxidase
  • mice response to the homologous antigen exhibited a higher immunological response as compared to the non-vaccinates.
  • the vaccinated mice When compared against a heterologous protein generated in the baculovirus platform, the vaccinated mice exhibited a limited response likely due to the baculovirus itself.
  • FIG. 1 shows specific immune response to Sapovirus protein.
  • FIG 2 shows Vaccinate vs Non-Vaccinate response to homologous protein.

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Abstract

La présente invention concerne des compositions immunogènes contenant des polypeptides immunogènes de sapovirus, comprenant des compositions immunogènes contenant des antigènes autres que des antigènes de sapovirus, comprenant des antigènes qui peuvent être utilisés dans l'immunisation contre des agents pathogènes qui provoquent des maladies diarrhéiques. L'invention divulgue également des méthodes de déclenchement d'une réponse immunitaire avec les compositions immunogènes ainsi que des méthodes de traitement d'une infection à sapovirus.
EP22834024.6A 2021-06-28 2022-06-28 Vaccins contre les sapovirus Pending EP4362975A2 (fr)

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